Compositions and Methods for Detecting C1orf43 Nucleic Acid

ABSTRACT

This disclosure provides oligomers, combinations of oligomers, compositions, kits, uses, and methods for detecting a C1orf43 nucleic acid, such as C1orf43 mRNA, such as human C1orf43 mRNA, in a sample.

This disclosure relates to oligomers, compositions, kits, and methodsuseful for the detection of C1orf43 nucleic acid.

SEQUENCE LISTING

The present application is filed with an electronically-formattedSequence Listing. The Sequence Listing is provided as a file entitled“2018-11-02_01159-0030-00PCT_Seq_List_ST25.txt” created on Nov. 2, 2018,which is 24,734 bytes in size. The information in theelectronically-formatted sequence listing is incorporated herein byreference in its entirety.

INTRODUCTION AND SUMMARY

Nucleic acid-based testing is an increasingly important approach fordetecting pathological or pre-pathological states such as infections,genetic abnormalities, and aberrant expression. Many tests useamplification of one or more target nucleic acids as a primarymechanism, where the presence or absence of the product(s) of theamplification (“amplicon(s)”) indicates the presence or absence of agiven state. The accuracy of such tests can depend on reliable controls,particularly in the case where no target product is observed. A reliablecontrol can establish that steps up to and including the amplificationand detection steps were performed without error, reducing thelikelihood of false negative results.

Ideally, a control would validate as many steps of a given process aspossible. For example, the detection of a control amplicon generallyindicates that at least the amplification and detection steps wereperformed without error and that the reagents used for amplification anddetection were not compromised. Where the template nucleic acid for thecontrol amplicon is obtained from the sample itself, the presence of thecontrol amplicon further indicates that sample collection and isolationwere performed without error.

It is also desirable for a control to be widely applicable, to reducethe need to identify and validate a different control for individualtests. For example, a widely applicable control may use a templatenucleic acid that is present in sufficient quantity in many cell typeswith little variation in genotype or expression level.

It is furthermore desirable for the reagents used to amplify the controlto perform robustly with good sensitivity and specificity, so that thepresence or absence of the control amplicon can be interpreted withconfidence.

As described herein, oligomers, compositions, kits, and methods usefulfor the detection of endogenous C1orf43 nucleic acid have been developedthat can meet one or more of these needs, e.g., that can be used inassays performed on a wide variety of samples, that can validate assaysteps from sample isolation through detection, that can perform robustlywith good sensitivity and specificity, or that at least provide thepublic with a useful choice.

Accordingly, the following embodiments are provided. Embodiment 1 is acombination of oligomers comprising at least first and secondamplification oligomers, wherein the first and second amplificationoligomers are reverse and forward amplification oligomers, respectively;each comprise at least 10 nucleotides; and are configured tospecifically hybridize to first and second sites in the sequence of SEQID NO: 39 and generate an amplicon therefrom, respectively.

Embodiment 2 is a method of detecting the presence or absence of aC1orf43 nucleic acid in a sample, comprising:

-   contacting the sample with a combination of oligomers comprising at    least first and second amplification oligomers,-   performing a nucleic acid amplification reaction which produces at    least a first amplicon in the presence of the C1orf43 nucleic acid,-   and detecting the presence or absence of the first amplicon,-   wherein: the first amplicon is produced through extension of the    first and second amplification oligomers in the presence of the    C1orf43 nucleic acid; and-   wherein the first and second amplification oligomers are reverse and    forward amplification oligomers, respectively; each comprise at    least 10 nucleotides; and are configured to specifically hybridize    to first and second sites in the sequence of SEQ ID NO: 39,    respectively.

Embodiment 3 is the combination of embodiment 1 or method of embodiment2, wherein at least one of the amplification oligomers is apromoter-primer.

Embodiment 4 is the combination or method of any one of the precedingembodiments, wherein the first amplification oligomer is apromoter-primer.

Embodiment 5 is the combination or method of any one of embodiments 3-4,wherein the promoter-primer comprises a T7 promoter which is located 5′of a target-hybridizing sequence.

Embodiment 6 is the combination or method of embodiment 5, wherein theT7 promoter comprises the sequence of SEQ ID NO: 58.

Embodiment 7 is the combination or method of any one of the precedingembodiments, wherein the first and second amplification oligomer areconfigured to generate an amplicon comprising at least 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, or 144 nucleotides of the sequenceof SEQ ID NO: 40.

Embodiment 8 is the combination or method of embodiment 7, wherein thefirst and second amplification oligomer are configured to generate anamplicon comprising the sequence of SEQ ID NO: 41.

Embodiment 9 is the combination or method of embodiment 7, wherein thefirst and second amplification oligomer are configured to generate anamplicon comprising the sequence of SEQ ID NO: 42.

Embodiment 10 is the combination or method of any one of the precedingembodiments, wherein the first amplification oligomer is configured tospecifically hybridize to a first site in the complement of SEQ ID NO:52.

Embodiment 11 is the combination or method of embodiment 10, wherein thefirst site comprises the sequence of SEQ ID NO: 53.

Embodiment 12 is the combination or method of embodiment 10, wherein thefirst site comprises the sequence of SEQ ID NO: 54.

Embodiment 13 is the combination or method of any one of the precedingembodiments, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 27,optionally with up to two mismatches.

Embodiment 14 is the combination or method of any one of embodiments1-12, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 29,optionally with up to two mismatches.

Embodiment 15 is the combination or method of any one of embodiments1-12, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 31,optionally with up to two mismatches.

Embodiment 16 is the combination or method of any one of embodiments1-12, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 33,optionally with up to two mismatches.

Embodiment 17 is the combination or method of any one of the precedingembodiments, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 27,29, 31, or 33.

Embodiment 18 is the combination or method of any one of the precedingembodiments, wherein the first amplification oligomer comprises thesequence of SEQ ID NO: 26, 28, 30, or 32.

Embodiment 19 is the combination or method of any one of the precedingembodiments, wherein the second amplification oligomer is configured tospecifically hybridize to a second site in the complement of SEQ ID NO:46.

Embodiment 20 is the combination or method of embodiment 19, wherein thesecond site comprises the sequence of SEQ ID NO: 47.

Embodiment 21 is the combination or method of embodiment 19, wherein thesecond site comprises the sequence of SEQ ID NO: 48.

Embodiment 22 is the combination or method of any one of the precedingembodiments, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 34,optionally with up to two mismatches.

Embodiment 23 is the combination or method of any one of embodiments1-21, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 35,optionally with up to two mismatches.

Embodiment 24 is the combination or method of any one of embodiments1-21, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 36,optionally with up to two mismatches.

Embodiment 25 is the combination or method of any one of embodiments1-21, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 37,optionally with up to two mismatches.

Embodiment 26 is the combination or method of any one of embodiments1-21, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 38,optionally with up to two mismatches.

Embodiment 27 is the combination or method of any one of the precedingembodiments, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 34,35, 36, 37, or 38.

Embodiment 28 is the combination or method of any one of the precedingembodiments, wherein the combination further comprises at least oneprobe oligomer that comprises at least 10 nucleotides and is configuredto specifically hybridize to an amplicon produced from the first andsecond amplification oligomers.

Embodiment 29 is the combination or method of embodiment 28, wherein theprobe oligomer is configured to specifically hybridize to a detectionsite in a nucleic acid having the sequence of SEQ ID NO: 43.

Embodiment 30 is a probe oligomer that comprises at least 10 nucleotidesand is configured to specifically hybridize to a detection site in anucleic acid having the sequence of SEQ ID NO: 43.

Embodiment 31 is the combination, method, or probe oligomer of any oneof embodiments 28-30, wherein the probe oligomer is configured tospecifically hybridize to a detection site in the sequence of SEQ ID NO:44.

Embodiment 32 is the combination, method, or probe oligomer of any oneof embodiments 28-30, wherein the probe oligomer is configured tospecifically hybridize to a detection site in a nucleic acid having thesequence of SEQ ID NO: 45.

Embodiment 33 is the combination, method, or probe oligomer of any oneof embodiments 28-30, wherein the probe oligomer is configured tospecifically hybridize to a detection site in a nucleic acid having thesequence of SEQ ID NO: 49.

Embodiment 34 is the combination, method, or probe oligomer ofembodiment 33, wherein the probe oligomer comprises a target hybridizingsequence comprising the sequence of SEQ ID NO: 50.

Embodiment 35 is the combination, method, or probe oligomer ofembodiment 33, wherein the probe oligomer comprises a target hybridizingsequence comprising the sequence of SEQ ID NO: 51.

Embodiment 36 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 15 with up totwo mismatches.

Embodiment 37 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 17 with up totwo mismatches.

Embodiment 38 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 19 with up totwo mismatches.

Embodiment 39 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 21 with up totwo mismatches.

Embodiment 40 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 23 with up totwo mismatches.

Embodiment 41 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 25 with up totwo mismatches.

Embodiment 42 is the combination, method, or probe oligomer of any oneof embodiments 28-41, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 15, 17, 19,21, 23, or 25.

Embodiment 43 is the combination, method, or probe oligomer of any oneof embodiments 28-41, wherein the probe oligomer comprises the sequenceof SEQ ID NO: 14, 16, 18, 20, 22, or 24.

Embodiment 44 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 59 with up totwo mismatches.

Embodiment 45 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 60 with up totwo mismatches.

Embodiment 46 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 64 with up totwo mismatches.

Embodiment 47 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 66 with up totwo mismatches.

Embodiment 48 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 67 with up totwo mismatches.

Embodiment 49 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 68 with up totwo mismatches.

Embodiment 50 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 69 with up totwo mismatches.

Embodiment 51 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 70 with up totwo mismatches.

Embodiment 52 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 71 with up totwo mismatches.

Embodiment 53 is the combination, method, or probe oligomer of any oneof embodiments 28-35, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 72 with up totwo mismatches.

Embodiment 54 is a probe oligomer that comprises a target hybridizingsequence comprising the sequence of any one of SEQ ID NO: 61, 62, 63, or65 with up to two mismatches

Embodiment 55 is the combination, method, or probe oligomer of any oneof embodiments 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 61 with up totwo mismatches.

Embodiment 56 is the combination, method, or probe oligomer of any oneof embodiments 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 62 with up totwo mismatches.

Embodiment 57 is the combination, method, or probe oligomer of any oneof embodiments 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 63 with up totwo mismatches.

Embodiment 58 is the combination, method, or probe oligomer of any oneof embodiments 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 65 with up totwo mismatches.

Embodiment 59 is the combination, method, or probe oligomer of any oneof embodiments 28-35 or 44-58, wherein the probe oligomer comprises atarget hybridizing sequence comprising the sequence of any one of SEQ IDNOs: 59-72.

Embodiment 60 is the combination, method, or probe oligomer of any oneof embodiments 28-59, wherein the probe oligomer comprises2′-O-methyl-ribose in its backbone.

Embodiment 61 is the combination, method, or probe oligomer ofembodiment 60, wherein at least half, at least 90%, or all of the sugarsin the probe oligomer are 2′-O-methyl-ribose.

Embodiment 62 is the combination or method of any one of embodiments1-27, wherein one or more of the first and second amplificationoligomers comprises a non-nucleotide detectable label.

Embodiment 63 is the combination, method, or probe oligomer of any oneof embodiments 28-61, wherein at least one probe oligomer comprises anon-nucleotide detectable label.

Embodiment 64 is the combination, method, or probe oligomer of any oneof embodiments 62-63, wherein the non-nucleotide detectable label is afluorescent label.

Embodiment 65 is the combination, method, or probe oligomer ofembodiment 63 or 64, wherein the probe oligomer comprises a quencher.

Embodiment 66 is the combination, method, or probe oligomer ofembodiment 65, wherein the non-nucleotide detectable label is afluorescent label and the quencher absorbs fluorescence to a greaterextent when the probe is free than when the probe is annealed to atarget nucleic acid.

Embodiment 67 is the combination, method, or probe oligomer of any oneof embodiments 64-66, wherein the fluorescent label is FAM, HEX, oracridine.

Embodiment 68 is the combination, method, or probe oligomer of any oneof embodiments 65-67, wherein the quencher is DABCYL or ROX.

Embodiment 69 is the combination, method, or probe oligomer of any oneof embodiments 65-68, wherein the fluorescent label is attached to the5′-terminus of the probe oligomer and the quencher is attached to the3′-terminus of the probe oligomer, or the fluorescent label is attachedto the 3′-terminus of the probe oligomer and the quencher is attached tothe 5′-terminus of the probe oligomer.

Embodiment 70 is the combination, method, or probe oligomer of any oneof embodiments 62-63, wherein the non-nucleotide detectable label is achemiluminescent label.

Embodiment 71 is the combination, method, or probe oligomer of any oneof embodiments 28-61 or 63-70, wherein the probe oligomer comprises afirst self-complementary region at its 5′ end and a secondself-complementary region at its 3′ end.

Embodiment 72 is the combination, method, or probe oligomer ofembodiment 71, wherein the self-complementary regions can hybridize toform 3 to 7 Watson-Crick or wobble base pairs.

Embodiment 73 is the combination, method, or probe oligomer ofembodiment 71, wherein the self-complementary regions can hybridize toform 4 Watson-Crick or wobble base pairs.

Embodiment 74 is the combination, method, or probe oligomer of any oneof embodiments 63-71, wherein the probe oligomer is a linear probeoligomer.

Embodiment 75 is a method of detecting the presence or absence of aC1orf43 nucleic acid in a sample, comprising:

-   contacting the sample with the probe oligomer of any one of    embodiments 30-61 or 63-74; performing a hybridization reaction    which produces a complex of the probe oligomer and the C1orf43    nucleic acid in the presence of the C1orf43 nucleic acid;-   and detecting the presence or absence of the complex of the probe    oligomer and the C1orf43 nucleic acid.

Embodiment 76 is the method of embodiment 75, wherein the hybridizationreaction is a hybridization protection assay.

Embodiment 77 is the method of embodiment 75 or 76, wherein thehybridization reaction is a dual kinetic assay.

Embodiment 78 is the method of any one of embodiments 75-77, wherein theprobe oligomer functions as a flasher probe in the dual kinetic assay.

Embodiment 79 is the method of any one of embodiments 75-77, wherein theprobe oligomer functions as a glower probe in the dual kinetic assay.

Embodiment 80 is the method of any one of embodiments 75-79, wherein theC1orf43 nucleic acid comprises a C1orf43 amplicon.

Embodiment 81 is the method of any one of embodiments 75-79, wherein theC1orf43 nucleic acid comprises C1orf43 RNA from cells in the sample.

Embodiment 82 is the method of any one of embodiments 75-81, wherein thesample is contacted with a combination of oligomers comprising the probeoligomer.

Embodiment 83 is the combination or method of any one of embodiments1-29, 31-56, or 82, wherein the combination further comprises at leastone capture oligomer that comprises at least 10 nucleotides and isconfigured to specifically hybridize to a capture site in a C1orf43nucleic acid.

Embodiment 84 is the combination or method of embodiment 83, wherein thecapture site is in the sequence of SEQ ID NO: 1, 2, or 3.

Embodiment 85 is the combination or method of embodiment 83, wherein thecapture site is in the sequence of SEQ ID NO: 39.

Embodiment 86 is a method of isolating C1orf43 nucleic acid from asample, comprising:

-   contacting the sample with at least one capture oligomer under    conditions permissive for forming one or more complexes of a capture    oligomer and the C1orf43 nucleic acid, thereby forming a    composition, wherein the capture oligomer comprises at least 10    nucleotides and is configured to specifically hybridize to a capture    site in the sequence of SEQ ID NO: 39; and isolating the capture    oligomer from the composition.

Embodiment 87 is the method of embodiment 86, wherein isolating thecapture oligomers comprises associating the capture oligomers with asolid support, and washing the solid support.

Embodiment 88 is the method of embodiment 87, wherein the solid supportcomprises a poly-N sequence that is complementary to a portion of thecapture oligomer.

Embodiment 89 is the method of embodiment 87, wherein the solid supportcomprises a binding agent that recognizes an affinity tag present in thecapture oligomer.

Embodiment 90 is a capture oligomer that comprises at least 10nucleotides and is configured to specifically hybridize to a capturesite in the sequence of SEQ ID NO: 39.

Embodiment 91 is the combination, method, or capture oligomer of any oneof embodiments 83-90, wherein the capture site is in the sequence of SEQID NO: 55.

Embodiment 92 is the combination, method, or capture oligomer of any oneof embodiments 83-90, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 56.

Embodiment 93 is the combination, method, or capture oligomer of any oneof embodiments 83-90, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 57.

Embodiment 94 is the combination, method, or capture oligomer of any oneof embodiments 83-93, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 5 with up totwo mismatches.

Embodiment 95 is the combination, method, or capture oligomer of any oneof embodiments 83-93, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 7 with up totwo mismatches.

Embodiment 96 is the combination, method, or capture oligomer of any oneof embodiments 83-93, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 9 with up totwo mismatches.

Embodiment 97 is the combination, method, or capture oligomer of any oneof embodiments 83-93, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 11 with up totwo mismatches.

Embodiment 98 is the combination, method, or capture oligomer of any oneof embodiments 83-93, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 13 with up totwo mismatches.

Embodiment 99 is the combination, method, or capture oligomer of any oneof embodiments 83-93, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 5, 7, 9, 11,or 13.

Embodiment 100 is the combination, method, or capture oligomer of anyone of embodiments 83-99, wherein the capture oligomer comprises thesequence of SEQ ID NO: 4, 6, 8, 10, or 12.

Embodiment 101 is the combination, method, or capture oligomer of anyone of embodiments 83-100, wherein the capture oligomer comprises2′-O-methyl-ribose in its backbone.

Embodiment 102 is the combination, method, or capture oligomer ofembodiment 101, wherein at least half, at least 90%, or all of thesugars in the target hybridizing sequence of the capture oligomer are2′-O-methyl-ribose.

Embodiment 103 is the combination, method, or capture oligomer of anyone of embodiments 83-102, wherein the capture oligomer furthercomprises a non-nucleotide affinity label.

Embodiment 104 is the combination, method, or capture oligomer of anyone of embodiments 83-102, wherein the capture oligomer furthercomprises a non-C1orf43 sequence.

Embodiment 105 is the combination, method, or capture oligomer ofembodiment 104, wherein the non-C1orf43 sequence is a poly-N sequence.

Embodiment 106 is the combination, method, or capture oligomer ofembodiment 105, wherein the poly-N sequence is a poly-A or poly-Tsequence.

Embodiment 107 is a combination comprising the capture oligomeraccording to any one of embodiments 90-106 and one or more amplificationoligomers, wherein the amplification oligomer is configured tospecifically hybridize to a site in the sequence of SEQ ID NO: 39.

Embodiment 108 is the combination of embodiment 107, wherein the one ormore amplification oligomers includes the first amplification oligomeras recited in any one of embodiments 4-18.

Embodiment 109 is the combination of embodiment 107 or 108, wherein theone or more amplification oligomers includes the second amplificationoligomer as recited in any one of embodiments 19-27.

Embodiment 110 is the combination of any one of embodiments 107-109,further comprising the probe oligomer as recited in any one ofembodiments 28-61 or 63-74.

Embodiment 111 is the method of any one of embodiments 86-89 or 91-106,further comprising performing a linear amplification wherein at leastone amplification oligomer is extended.

Embodiment 112 is the method of embodiment 111, wherein prior to thelinear amplification, the amplification oligomer is associated with acomplex of C1orf43 nucleic acid and a capture oligomer and the complexis associated with a solid support, and the method comprises washing thesolid support.

Embodiment 113 is the method of embodiment 112, wherein the solidsupport is a population of microbeads.

Embodiment 114 is the method of embodiment 113, wherein the microbeadsof the population are magnetic.

Embodiment 115 is the method of any one of embodiments 112-114, whereinfollowing the washing step, the method comprises adding one or moreadditional amplification oligomers oppositely oriented to anamplification oligomer associated with the complex of C1orf43 nucleicacid and the capture oligomer.

Embodiment 116 is the method of embodiment 115, wherein the one or moreoppositely oriented additional amplification oligomers includes apromoter-primer.

Embodiment 117 is the method of embodiment 116, wherein the one or moreoppositely oriented additional amplification oligomers includes anoligomer that is not a promoter-primer.

Embodiment 118 is the method of any one of embodiments 115-117, whereinthe one or more oppositely oriented additional amplification oligomersincludes the second amplification oligomer as recited in any one ofembodiments 19-27.

Embodiment 119 is the method of any one of embodiments 115-118, furthercomprising performing an exponential amplification following the linearamplification.

Embodiment 120 is the method of embodiment 119, wherein the exponentialamplification is transcription-mediated amplification.

Embodiment 121 is the method of any one of embodiments 2-29, 31-89,91-105, or 111-120, further comprising quantifying C1orf43 nucleic acidin the sample.

Embodiment 122 is a kit or composition comprising at least one, two,three, or four of a first amplification oligomer, a second amplificationoligomer, a probe oligomer, or a capture oligomer recited in any one ofthe preceding embodiments.

Embodiment 123 is the kit or composition of embodiment 122, comprisingat least one probe oligomer as recited in any one of embodiments 28-61or 63-74.

Embodiment 124 is the kit or composition of any one of embodiments122-123, comprising at least one capture oligomer as recited in any oneof embodiments 83-85 or 90-106.

Embodiment 125 is the kit or composition of any one of embodiments122-124, comprising the first amplification oligomer as recited in anyone of embodiments 4-18 and the second amplification oligomer as recitedin any one of embodiments 19-27.

Embodiment 126 is a kit according to any one of embodiments 122-125 orcomprising the combination of any one of embodiments 1, 3-62, 63-85, or91-110.

Embodiment 127 is a composition according to any one of embodiments122-125 or comprising the combination of any one of embodiments 1, 3-62,63-85, or 91-110.

Embodiment 128 is the composition of embodiment 127, which is aqueous,frozen, or lyophilized.

Embodiment 129 is the use of the combination, method, composition,capture oligomer, probe oligomer, or kit of any one of the precedingembodiments for detecting the presence or absence of a C1orf43 nucleicacid in a sample.

Embodiment 130 is the combination, method, composition, captureoligomer, probe oligomer, or kit of any one of embodiments 1-128, foruse in detecting the presence or absence of a C1orf43 nucleic acid in asample.

Embodiment 131 is the use of the combination, method, composition,capture oligomer, probe oligomer, or kit of any one of embodiments 1-128for quantifying a C1orf43 nucleic acid in a sample.

Embodiment 132 is the combination, method, composition, captureoligomer, probe oligomer, or kit of any one of embodiments 1-128, foruse in quantifying a C1orf43 nucleic acid in a sample.

Embodiment 133 is the use, combination, method, composition, captureoligomer, probe oligomer, or kit of any one of embodiments 2-29, 31-53,55-89, 91-106, 111-121, or 126-132, wherein the sample comprises humanmRNA.

Embodiment 134 is the use, combination, method, composition, or kit ofembodiment 133, wherein the human mRNA comprises mRNA from bladder,ductus deferens, epididymis, kidney, lymph node, pancreas, peripheralblood lymphocytes, penis, prostate, seminal vesicle, or spleen.

Embodiment 135 is the use, combination, method, composition, or kit ofembodiment 133, wherein the human mRNA comprises mRNA from a vaginal orcervical sample.

Embodiment 136 is the use, combination, method, composition, or kit ofembodiment 135, wherein the vaginal or cervical sample is a vaginal orcervical swab.

Embodiment 137 is the use, combination, method, composition, or kit ofany one of embodiments 2-29, 31-53, 55-89, 91-106, 111-121, or 126-136,wherein the method or use further comprises detecting the presence orabsence of at least one nucleic acid of a microbe or pathogen.

Embodiment 138 is the use, combination, method, composition, or kit ofembodiment 137, wherein the at least one nucleic acid of a microbe orpathogen comprises human papillomavirus nucleic acid, Chlamydiatrachomatis nucleic acid, Neisseria gonorrheae nucleic acid, Trichomonasvaginalis nucleic acid, or Mycoplasma genitalium nucleic acid.

Embodiment 139 is the use, combination, method, composition, or kit ofany one of embodiments 2-29, 31-53, 55-89, 91-106, 111-121, or 126-138,wherein the method or use further comprises detecting the presence orabsence of at least one mRNA other than C1orf43.

Embodiment 140 is the use, combination, method, composition, or kit ofembodiment 139, wherein the mRNA other than C1orf43 is a human mRNAother than C1orf43.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS A. Overview

As demonstrated below in the examples, it has been found that C1orf43 isexpressed in a wide variety of tissues, and oligomers have beendeveloped that can detect and quantify C1orf43 in a sensitive andspecific manner. Accordingly, this disclosure provides oligomers,compositions, kits, and methods useful for the detection of C1orf43nucleic acid. Detection of C1orf43 can be used as a positive control incombination with nucleic acid assays on samples that should containC1orf43 nucleic acid (e.g., C1orf43 mRNA), such as samples that shouldcontain mammalian, primate, or human tissue or cells. Detection ofC1orf43 can validate such assays by allowing a negative result for ananalyte of interest to be interpreted with confidence as a true negativeresult and not a false negative resulting from a failure in one or moreof sample acquisition, nucleic acid isolation, amplification, and/orprobe detection.

Thus, for example, embodiments provided herein include a combination ofat least two amplification oligomers for amplifying a C1orf43 ampliconwithin or comprising the region of C1orf43 shown as SEQ ID NO: 39 and adetection oligomer for detecting the C1orf43 amplicon is provided, whichoptionally further comprises at least one capture oligomer for isolatingC1orf43 nucleic acid from a sample. In a further embodiment, theamplification oligomers are for an amplification reaction that beginswith C1orf43 mRNA, for example, transcription-mediated amplification.

B. Definitions

Before describing the present teachings in detail, it is to beunderstood that the disclosure is not limited to specific compositionsor process steps, as such may vary. Measured and measureable values areunderstood to be approximate, taking into account significant digits andthe error associated with the measurement. It should be noted that, asused in this specification and the appended claims, the singular form“a”, “an” and “the” include plural references unless the context clearlydictates otherwise. Thus, for example, reference to “an oligomer”includes a plurality of oligomers and the like.

The terms “comprise”, “comprises”, “comprising”, “contain”, “contains”,“containing”, “include”, “includes”, and “including” are open-ended andnot intended to be limiting. Unless specifically noted, embodiments inthe specification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims).

It is to be understood that both the foregoing general description anddetailed description are exemplary and explanatory only and are notrestrictive of the teachings. To the extent that any materialincorporated by reference is inconsistent with the express content ofthis disclosure, the express content controls. Section headings areprovided for the convenience of the reader and do not limit the scope ofthe disclosure.

“Sample” includes any specimen (e.g., biological, environmental,synthetic, or forensic) that may contain C1orf43 nucleic acid.“Biological samples” include any tissue or material derived from aliving or dead subject (e.g., mammal, primate, or human; alsocontemplated are transgenic cells or organisms comprising C1orf43nucleic acid) that may contain C1orf43 nucleic acid, including, e.g.,peripheral blood, plasma, serum, bladder, ductus deferens, epididymis,kidney, lymph node, pancreas, peripheral blood lymphocytes, penis,prostate, seminal vesicle, spleen, vaginal or cervical samples (e.g.,swabs), or other body fluids or materials. The biological sample may betreated to physically or mechanically disrupt tissue or cell structure,thus releasing intracellular components into a solution which mayfurther contain enzymes, buffers, salts, detergents and the like, whichare used to prepare a biological sample for analysis. Also, samples mayinclude processed samples, such as those obtained from passing samplesover or through a filtering device, or following centrifugation, or byadherence to a medium, matrix, or support.

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see, e.g., International PatentApplication Pub. No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties ofthe nucleic acid may be either ribose or deoxyribose, or similarcompounds having known substitutions such as, for example, 2′-methoxysubstitutions and 2′-halide substitutions (e.g., 2′-F). Nitrogenousbases may be conventional bases (A, G, C, T, U), analogs thereof (e.g.,inosine, 5-methylisocytosine, isoguanine; see, e.g., The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham etal., 2007, BioTechniques 43: 617-24), which include derivatives ofpurine or pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidine bases havingsubstituent groups at the 5 or 6 position, purine bases having analtered or replacement substituent at the 2, 6 and/or 8 position, suchas 2-amino-6-methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO⁴-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or3-substituted pyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825,6,949,367 and International Patent Application Pub. No. WO 93/13121,each incorporated by reference herein). Nucleic acids may include“abasic” residues in which the backbone does not include a nitrogenousbase for one or more residues (see. e.g., U.S. Pat. No. 5,585,481,incorporated by reference herein). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′-methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., Biochemistry 43:13233-41, 2004, incorporated by reference herein).Nucleic acids may include modified bases to alter the function orbehavior of the nucleic acid, e.g., addition of a 3′-terminaldideoxynucleotide to block additional nucleotides from being added tothe nucleic acid. Synthetic methods for making nucleic acids in vitroare well-known in the art although nucleic acids may be purified fromnatural sources using routine techniques.

A “C1orf43 nucleic acid” is a nucleic acid that occurs in, is at least90% or at least 95% identical to, or contains no more than one mismatchrelative to any allele of C1orf43, such that, for example, “14contiguous nucleotides of C1orf43 nucleic acid sequence” refers to a14-mer that matches at least 13 out of 14 positions of an allele ofC1orf43 (including the coding strand or the complement thereof). Thepresence of a U is considered equivalent to a T and vice versa forpurposes of determining whether a sequence qualifies as a C1orf43nucleic acid sequence. The target-hybridizing regions of exemplaryoligomers disclosed herein, the C1orf43-derived sequence of in vitrotranscripts disclosed herein, and subsequences thereof are alsoconsidered C1orf43 nucleic sequence. Thus, examples of C1orf43 sequenceinclude SEQ ID NOs: 1-3, 39-57, and any sequences identified herein astarget-hybridizing sequences, and complements thereof. Percent identitycan be determined using an appropriate alignment algorithm such as theNeedleman-Wunsch algorithm with standard parameters.

The term “polynucleotide” as used herein denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Synthetic nucleic acids, e.g., DNA, RNA,DNA/RNA chimerics, (including when non-natural nucleotides or analoguesare included therein), are typically synthesized “3′-to-5′,” i.e., bythe addition of nucleotides to the 5′-terminus of a growing nucleicacid. A polynucleotide or oligomer is considered to comprise two (ormore) specified SEQ ID NOs if each of the sequence of the SEQ ID NOs ispresent, regardless of whether they overlap. Thus, as a simplifiedexample, the sequence CAT comprises both CA and AT.

A “nucleotide” as used herein is a subunit of a nucleic acid consistingof a phosphate group, a 5-carbon sugar, and a nitrogenous base (alsoreferred to herein as “nucleobase”). The 5-carbon sugar found in RNA isribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The term alsoincludes analogs of such subunits, such as a methoxy group at the 2′position of the ribose (also referred to herein as “2′-O-Me” or“2′-methoxy”). As used herein, methoxy oligonucleotides containing “T”residues have a methoxy group at the 2′ position of the ribose moiety,and a uracil at the base position of the nucleotide.

A “non-nucleotide unit” as used herein is a unit that does notsignificantly participate in hybridization of a polymer. Such units donot, for example, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” as used herein is a nucleic acid comprising atarget sequence to be amplified. Target nucleic acids may be DNA or RNAas described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence, which may not be amplified.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid that is to be amplifiedand/or detected. The “target sequence” includes the complexing sequencesto which oligonucleotides (e.g., priming oligonucleotides and/orpromoter oligonucleotides) complex during an amplification processes(e.g., TMA). Where the target nucleic acid is originallysingle-stranded, the term “target sequence” will also refer to thesequence complementary to the “target sequence” as present in the targetnucleic acid. Where the target nucleic acid is originallydouble-stranded, the term “target sequence” refers to both the sense orcoding (+) and antisense or template (−) strands. The (+) strandcorresponds to the mRNA sequence and the (−) is the complement thereof.The exemplary sequence of SEQ ID NO: 1 represents a (+) strand.

“Target-hybridizing sequence” is used herein to refer to the portion ofan oligomer that is configured to hybridize with a target sequence. Insome embodiments, the target-hybridizing sequences are configured tospecifically hybridize with a target nucleic acid sequence.Target-hybridizing sequences may be 100% complementary to the portion ofthe target sequence to which they are configured to hybridize, but notnecessarily. Target-hybridizing sequences may also include inserted,deleted and/or substituted nucleotide residues relative to a targetsequence. Less than 100% complementarity of a target-hybridizingsequence to a target sequence may arise, for example, when the targetnucleic acid comprises one or more polymorphic positions (e.g., SNPs),such as would be the case for an oligomer configured to hybridize tovarious alleles of C1orf43. It is understood that other reasons existfor configuring a target-hybridizing sequence to have less than 100%complementarity to a target nucleic acid.

The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of a referenced oligonucleotidetarget-hybridizing sequence. For example, amplification oligomers thatare “configured to generate an amplicon” have polynucleotide sequencesthat hybridize to the target sequence and can be used in anamplification reaction to generate the amplicon; for example, in thecontext of PCR or TMA reactions, the oligomers have hybridization sitesbounding at least one nucleotide and their 3′ ends are properly orientedfor amplification when hybridized.

The term “configured to specifically hybridize to” as used herein meansthat the target-hybridizing region of an amplification oligonucleotide,detection probe, or other oligonucleotide has a polynucleotide sequencethat could target a sequence of the referenced target region. Such anoligonucleotide is not limited to targeting that sequence only, but israther useful as a composition, in a kit, or in a method for targetingthe target nucleic acid. The oligonucleotide can function as a componentof an assay for amplification and detection of C1orf43 from a sample,and therefore can target C1orf43 in the presence of other nucleic acidscommonly found in testing samples. “Specifically hybridize to” does notmean exclusively hybridize to, as some small level of hybridization tonon-target nucleic acids may occur, as is understood in the art. Rather,“specifically hybridize to” means that the oligonucleotide is configuredto function in an assay to primarily hybridize the target so that anaccurate detection of target nucleic acid in a sample can be determined.Unless the context indicates otherwise, an oligomer is considered tospecifically hybridize to a given sequence if it specifically hybridizesto either strand of a double-stranded version of that sequence.

The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of 5 ntresidues and an upper limit of 500 to 900 nt residues. In someembodiments, oligonucleotides are in a size range having a lower limitof 12 to 15 nt and an upper limit of 50 to 600 nt, and other embodimentsare in a range having a lower limit of 15 to 20 nt and an upper limit of22 to 100 nt. Oligonucleotides may be purified from naturally occurringsources or may be synthesized using any of a variety of well-knownenzymatic or chemical methods. The term oligonucleotide does not denoteany particular function to the reagent; rather, it is used genericallyto cover all such reagents described herein. An oligonucleotide mayserve various different functions. For example, it may function as aprimer if it is specific for and capable of hybridizing to acomplementary strand and can further be extended in the presence of anucleic acid polymerase; it may function as a primer and provide apromoter if it contains a sequence recognized by an RNA polymerase andallows for transcription (e.g., a T7 Primer); and it may function todetect a target nucleic acid if it is capable of hybridizing to thetarget nucleic acid, or an amplicon thereof, and further provides adetectible moiety (e.g., a fluorophore).

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. Oligomersnot intended for extension by a nucleic acid polymerase may include ablocker group that replaces the 3′ OH to prevent enzyme-mediatedextension of the oligomer in an amplification reaction. For example,blocked amplification oligomers and/or detection probes present duringamplification may not have functional 3′ OH and instead include one ormore blocking groups located at or near the 3′ end. In some embodiments,a blocking group near the 3′ end and may be within five residues of the3′ end and is sufficiently large to limit binding of a polymerase to theoligomer. In other embodiments, a blocking group is covalently attachedto the 3′ terminus. Many different chemical groups may be used to blockthe 3′ end, e.g., alkyl or substituted alkyl groups (e.g., hexanediol),non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid, and which hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. An example of an amplification oligomer isa “primer” that hybridizes to a target nucleic acid and contains a 3′ OHend that is extended by a polymerase in an amplification process. Insome embodiments, the 5′ region of an amplification oligonucleotide mayinclude a promoter sequence that is non-complementary to the targetnucleic acid (which may be referred to as a “promoter primer”). Anotherexample of an amplification oligomer is an oligomer that is not extendedby a polymerase (e.g., because it has a 3′ blocked end) but participatesin or facilitates amplification. For example, the 5′ region of anamplification oligonucleotide may include a promoter sequence that isnon-complementary to the target nucleic acid (which may be referred toas a “promoter provider”). Those skilled in the art will understand thatan amplification oligomer that functions as a primer may be modified toinclude a 5′ promoter sequence, and thus function as a promoter primer.Incorporating a 3′ blocked end further modifies the promoter primer,which is now capable of hybridizing to a target nucleic acid andproviding an upstream promoter sequence that serves to initiatetranscription, but does not provide a primer for oligo extension. Such amodified oligo is referred to herein as a “promoter provider” oligomer.Size ranges for amplification oligonucleotides include those that are 10to 70 nt long (not including any promoter sequence or poly-A tails) andcontain at least 10 contiguous bases, or even at least 12 contiguousbases that are complementary to a region of the target nucleic acidsequence (or a complementary strand thereof). The contiguous bases areat least 80%, or at least 90%, or completely complementary to the targetsequence to which the amplification oligomer binds. An amplificationoligomer may optionally include modified nucleotides or analogs, oradditional nucleotides that participate in an amplification reaction butare not complementary to or contained in the target nucleic acid, ortemplate sequence. It is understood that when referring to ranges forthe length of an oligonucleotide, amplicon, or other nucleic acid, thatthe range is inclusive of all whole numbers (e.g., 19-25 contiguousnucleotides in length includes 19, 20, 21, 22, 23, 24 & 25).

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (see. e.g., U.S. Pat. No.4,786,600, incorporated by reference herein). PCR amplification uses aDNA polymerase, pairs of primers, and thermal cycling to synthesizemultiple copies of two complementary strands of dsDNA or from a cDNA(see. e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159; eachincorporated by reference herein). LCR amplification uses four or moredifferent oligonucleotides to amplify a target and its complementarystrand by using multiple cycles of hybridization, ligation, anddenaturation (see. e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663, eachincorporated by reference herein). SDA uses a primer that contains arecognition site for a restriction endonuclease and an endonuclease thatnicks one strand of a hemimodified DNA duplex that includes the targetsequence, whereby amplification occurs in a series of primer extensionand strand displacement steps (see. e.g., U.S. Pat. Nos. 5,422,252;5,547,861; and 5,648,211; each incorporated by reference herein).

As used herein, the term “linear amplification” refers to anamplification mechanism that is designed to produce an increase in thetarget nucleic acid linearly proportional to the amount of targetnucleic acid in the reaction. For instance, multiple RNA copies can bemade from a DNA target using a transcription-associated reaction, wherethe increase in the number of copies can be described by a linear factor(e.g., starting copies of template×100). In some embodiments, a firstphase linear amplification in a multiphase amplification procedureincreases the starting number of target nucleic acid strands or thecomplements thereof by at least 10 fold, e.g., by at least 100 fold, orby 10 to 1,000 fold before the second phase amplification reaction isbegun. An example of a linear amplification system is “T7-based LinearAmplification of DNA” (TLAD; see Liu et al., BMC Genomics, 4: Art. No.19, May 9, 2003). Other methods are known, e.g., from U.S. Pat. No.9,139,870, or disclosed herein. Accordingly, the term “linearamplification” refers to an amplification reaction which does not resultin the exponential amplification of a target nucleic acid sequence. Theterm “linear amplification” does not refer to a method that simply makesa single copy of a nucleic acid strand, such as the transcription of anRNA molecule into a single cDNA molecule as in the first-strandsynthesis step of reverse transcription (RT)-PCR.

As used herein, the term “exponential amplification” refers to nucleicacid amplification that is designed to produce an increase in the targetnucleic acid geometrically proportional to the amount of target nucleicacid in the reaction. For example, PCR produces one DNA strand for everyoriginal target strand and for every synthesized strand present.Similarly, transcription-associated amplification produces multiple RNAtranscripts for every original target strand and for every subsequentlysynthesized strand. The amplification is exponential because thesynthesized strands are used as templates in subsequent rounds ofamplification. An amplification reaction need not actually produceexponentially increasing amounts of nucleic acid to be consideredexponential amplification, so long as the amplification reaction isdesigned to produce such increases.

“Transcription-associated amplification” or “transcription-mediatedamplification” (TMA) refer to nucleic acid amplification that uses anRNA polymerase to produce multiple RNA transcripts from a nucleic acidtemplate. These methods generally employ an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a template complementary oligonucleotide thatincludes a promoter sequence, e.g., a T7 promoter, and optionally mayinclude one or more other oligonucleotides. When a T7promoter-containing oligomer is used, it may be referred to as a “T7primer” or “T7 oligomer”; other primers/oligomers may be referred to as“non-T7” or “NT7” primers/oligomers. TMA methods and single-primertranscription-associated amplification methods are embodiments ofamplification methods used for detection of C1orf43 target sequences asdescribed herein. Variations of transcription-associated amplificationare well-known in the art as previously disclosed in detail (see. e.g.,U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990;5,554,516; and 7,374,885; and International Patent Application Pub. Nos.WO 88/01302; WO 88/10315; and WO 95/03430; each incorporated byreference herein). The person of ordinary skill in the art willappreciate that the disclosed compositions may be used in amplificationmethods based on extension of oligomer sequences by a polymerase.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored through real-time detection.

The term “amplicon” or “amplification product” as used herein refers tothe nucleic acid molecule generated during an amplification procedurethat is complementary or homologous to a sequence contained within thetarget sequence. The complementary or homologous sequence of an ampliconis sometimes referred to herein as a “target-specific sequence.”Amplicons generated using the amplification oligomers of the currentdisclosure may comprise non-target specific sequences. Amplicons can bedouble-stranded or single-stranded and can include DNA, RNA, or both.For example, DNA-dependent RNA polymerase transcribes single-strandedamplicons from double-stranded DNA during transcription-mediatedamplification procedures. These single-stranded amplicons are RNAamplicons and can be either strand of a double-stranded complex,depending on how the amplification oligomers are configured. Thus,amplicons can be single-stranded RNA. RNA-dependent DNA polymerasessynthesize a DNA strand that is complementary to an RNA template. Thus,amplicons can be double-stranded DNA and RNA hybrids. RNA-dependent DNApolymerases often include RNase activity, or are used in conjunctionwith an RNase, which degrades the RNA strand. Thus, amplicons can besingle stranded DNA. RNA-dependent DNA polymerases and DNA-dependent DNApolymerases synthesize complementary DNA strands from DNA templates.Thus, amplicons can be double-stranded DNA. RNA-dependent RNApolymerases synthesize RNA from an RNA template. Thus, amplicons can bedouble-stranded RNA. DNA-dependent RNA polymerases synthesize RNA fromdouble-stranded DNA templates, also referred to as transcription. Thus,amplicons can be single stranded RNA. Amplicons and methods forgenerating amplicons are known to those skilled in the art. Forconvenience herein, a single strand of RNA or a single strand of DNA mayrepresent an amplicon generated by an amplification oligomer combinationof the current disclosure. Such representation is not meant to limit theamplicon to the representation shown. Skilled artisans in possession ofthe instant disclosure will use amplification oligomers and polymeraseenzymes to generate any of the numerous types of amplicons, all withinthe spirit and scope of the current disclosure.

“Detection probe,” “detection oligonucleotide,” “probe oligomer,” and“detection probe oligomer” are used interchangeably to refer to anucleic acid oligomer that hybridizes specifically to a target sequencein a nucleic acid, or in an amplified nucleic acid, under conditionsthat promote hybridization to allow detection of the target sequence oramplified nucleic acid. Detection may either be direct (e.g., a probehybridized directly to its target sequence) or indirect (e.g., a probelinked to its target via an intermediate molecular structure). Detectionprobes may be DNA, RNA, analogs thereof or combinations thereof (e.g.,DNA/RNA chimerics) and they may be labeled or unlabeled. Detectionprobes may further include alternative backbone linkages such as, e.g.,2′-O-methyl linkages. A detection probe's “target sequence” generallyrefers to a smaller nucleic acid sequence region within a larger nucleicacid sequence that hybridizes specifically to at least a portion of aprobe oligomer by standard base pairing. A detection probe may comprisetarget-specific sequences and other sequences that contribute to thethree-dimensional conformation of the probe (see. e.g., U.S. Pat. Nos.5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945;and US Patent Application Pub. No. 20060068417; each incorporated byreference herein).

As used herein, a “linear” detection probe, oligomer, or oligonucleotideis a detection oligomer that does not substantially form conformationsheld by intramolecular bonds, e.g., is configured to hybridize alongsubstantially all of its length to its target sequence and/or lacks aself-complementary segment of 3 or more nucleotides at or near its 5′and 3′ ends (such as the terminal 7, 6, 5, or 4 nucleotides do notcomprise three consecutive self-complementary nucleotides) that can forma hairpin or other self-hybridized secondary structure.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labeling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g., hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labelingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, antibody, or additional oligomer, which is either directlyor indirectly labeled, and which may amplify the detectable signal.Labels include any detectable moiety, such as a radionuclide, ligand(e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, orchromophore (e.g., dye, particle, or bead that imparts detectablecolor), luminescent compound (e.g., bioluminescent, phosphorescent, orchemiluminescent labels), or fluorophore. Labels may be detectable in ahomogeneous assay in which bound labeled probe in a mixture exhibits adetectable change different from that of an unbound labeled probe, e.g.,instability or differential degradation properties. A “homogeneousdetectable label” can be detected without physically removing bound fromunbound forms of the label or labeled probe (see. e.g., U.S. Pat. Nos.5,283,174; 5,656,207; and 5,658,737; each incorporated by referenceherein). Labels include chemiluminescent compounds, e.g., acridiniumester (“AE”) compounds that include standard AE and derivatives (see.e.g., U.S. Pat. Nos. 5,656,207; 5,658,737; and 5,639,604; eachincorporated by reference herein). Synthesis and methods of attachinglabels to nucleic acids and detecting labels are well known. (See. e.g.,Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd ed. (ColdSpring Harbor Laboratory Press, Cold Spring Habor, N Y, 1989), Chapter10, incorporated by reference herein. See also U.S. Pat. Nos. 5,658,737;5,656,207; 5,547,842; 5,283,174; and 4,581,333; each incorporated byreference herein). More than one label, and more than one type of label,may be present on a particular probe, or detection may use a mixture ofprobes in which each probe is labeled with a compound that produces adetectable signal (see. e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579,each incorporated by reference herein).

“Capture probe,” “capture oligonucleotide,” “capture oligomer,” “targetcapture oligomer,” and “capture probe oligomer” are used interchangeablyto refer to a nucleic acid oligomer that specifically hybridizes to atarget sequence in a target nucleic acid by standard base pairing andjoins to a binding partner on an immobilized probe to capture the targetnucleic acid to a support. One example of a capture oligomer includestwo binding regions: a sequence-binding region (e.g., target-specificportion) and an immobilized probe-binding region, usually on the sameoligomer, although the two regions may be present on two differentoligomers joined together by one or more linkers. Another embodiment ofa capture oligomer uses a target-sequence binding region that includesrandom or non-random poly-GU, poly-GT, or poly U sequences to bindnon-specifically to a target nucleic acid and link it to an immobilizedprobe on a support.

As used herein, an “immobilized oligonucleotide,” “immobilized probe,”“immobilized binding partner,” “immobilized oligomer,” or “immobilizednucleic acid” refers to a nucleic acid binding partner that joins acapture oligomer to a support, directly or indirectly. An immobilizedprobe joined to a support facilitates separation of a capture probebound target from unbound material in a sample. One embodiment of animmobilized probe is an oligomer joined to a support that facilitatesseparation of bound target sequence from unbound material in a sample.Supports may include known materials, such as matrices and particlesfree in solution, which may be made of nitrocellulose, nylon, glass,polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal,or other compositions, of which one embodiment is magneticallyattractable particles. Supports may be monodisperse magnetic spheres(e.g., uniform size+5%), to which an immobilized probe is joineddirectly (via covalent linkage, chelation, or ionic interaction), orindirectly (via one or more linkers), where the linkage or interactionbetween the probe and support is stable during hybridization conditions.

“Sample preparation” refers to any steps or method that treats a samplefor subsequent amplification and/or detection of nucleic acids presentin the sample. Samples may be complex mixtures of components of whichthe target nucleic acid is a minority component. Sample preparation mayinclude any known method of concentrating components, such as microbesor nucleic acids, from a larger sample volume, such as by filtration ofairborne or waterborne particles from a larger volume sample or byisolation of microbes from a sample by using standard microbiologymethods. Sample preparation may include physical disruption and/orchemical lysis of cellular components to release intracellularcomponents into a substantially aqueous or organic phase and removal ofdebris, such as by using filtration, centrifugation or adsorption.Sample preparation may include use of a nucleic acid oligonucleotidethat selectively or non-specifically capture a target nucleic acid andseparate it from other sample components (e.g., as described in U.S.Pat. No. 6,110,678 and International Patent Application Pub. No. WO2008/016988, each incorporated by reference herein).

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. “Separating” or“purifying” does not connote any degree of purification. Typically,separating or purifying removes at least 70%, or at least 80%, or atleast 95% of the target nucleic acid from other sample components.

As used herein, the terms “TTime,” “emergence time,” and “time ofemergence” are interchangeable and represent the threshold time or timeof emergence of signal in a real-time plot of the assay data. TTimevalues estimate the time at which a particular threshold indicatingamplicon production is passed in a real-time amplification reaction.TTime and an algorithm for calculating and using TTime values aredescribed in Light et al., U.S. Pub. No. 2006/0276972, paragraphs [0517]through [0538], the disclosure of which is incorporated by referenceherein. A curve fitting procedure is applied to normalized andbackground-adjusted data. The curve fit is performed for only a portionof the data between a predetermined low bound and high bound. The goal,after finding the curve that fits the data, is to estimate the timecorresponding to the point at which the curve or a projection thereofintersects a predefined threshold value. In one embodiment, thethreshold for normalized data is 0.11. The high and low bounds aredetermined empirically as that range over which curves fit to a varietyof control data sets exhibit the least variability in the timeassociated with the given threshold value. For example, in oneembodiment, the low bound is 0.04 and the high bound is 0.36. The curveis fit for data extending from the first data point below the low boundthrough the first data point past the high bound. Next, there is made adetermination whether the slope of the fit is statistically significant.For example, if thep value of the first order coefficient is less than0.05, the fit is considered significant, and processing continues. Ifnot, processing stops. Alternatively, the validity of the data can bedetermined by the R² value. The slope m and intercept b of the linearcurve y=mx+b are determined for the fitted curve. With that information,TTime can be determined as follows: TTime=(Threshold−b)/m.

References, particularly in the claims, to “the sequence of SEQ ID NO:X” refer to the base sequence of the corresponding sequence listingentry and do not require identity of the backbone (e.g., RNA, 2′-O-MeRNA, or DNA) unless otherwise indicated. Furthermore, T and U residuesare to be considered interchangeable for purposes of sequence listingentries unless otherwise indicated, e.g., a sequence can be consideredidentical to SEQ ID NO: 6 regardless of whether the residue at theseventh position is a T or a U.

C. Oligomers, Compositions, and Kits

The present disclosure provides oligomers, compositions, and kits,useful for amplifying, detecting, or quantifying C1orf43 from a sample.

In some embodiments, amplification oligomers are provided. Amplificationoligomers generally comprise a target-hybridizing region, e.g.,configured to hybridize specifically to a C1orf43 nucleic acid. Whileoligomers of different lengths and base composition may be used foramplifying C1orf43 nucleic acids, in some embodiments oligomers in thisdisclosure have target-hybridizing regions from 10 to 60 bases inlength, from 14 to 50 bases in length, or from 15 to 40 bases in length.

In certain embodiments, an amplification oligomer as described herein isa promoter primer further comprising a promoter sequence located 5′ tothe target-hybridizing sequence and which is non-complementary to theC1orf43 target nucleic acid. For example, in some embodiments of anoligomer combination as described herein for amplification of a C1orf43target region, an amplification oligomer as described above is apromoter primer further comprising a promoter sequence 5′ to thetarget-hybridizing sequence. Alternatively, an amplification oligomercan be a promoter provider comprising a promoter sequence. In particularembodiments, the promoter sequence is a T7 RNA polymerase promotersequence such as, for example, a T7 promoter sequence having thesequence shown in SEQ ID NO: 58. In some embodiments, at least one,e.g., two, three, or four promoter primers are provided comprising atarget-hybridizing sequence that hybridizes to (+)-strand (codingstrand) C1orf43 sequence.

Exemplary target-hybridizing sequences that hybridize to (+)-strand(coding strand) C1orf43 sequence are SEQ ID NOs: 27, 29, 31, and 33.Exemplary promoter-primers that hybridize to (+)-strand (coding strand)C1orf43 sequence are SEQ ID NOs: 26, 28, 30, and 32.

In some embodiments, an amplification oligomer is not a promoter primeror does not comprise a promoter sequence. For example, in PCR-basedapproaches the primers are generally not promoter primers, and inTMA-based approaches at least one primer that is not a promoter primeris typically used (while at least one promoter primer is also used). Insome embodiments, at least one, e.g., two, three, or four amplificationoligomers that are not promoter primers are provided comprising atarget-hybridizing sequence that hybridizes to (−)-strand (templatestrand) C1orf43 sequence.

Exemplary target-hybridizing sequences that hybridize to (−)-strand(template strand) C1orf43 sequence are SEQ ID NOs: 34, 35, 36, 37, and38.

The amplification oligomers discussed above can be used in anamplification reaction configured to produce an amplicon, e.g.,comprising C1orf43 sequence such as at least 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, or 144 nucleotides of the sequence of SEQ IDNO: 40; the sequence of SEQ ID NO: 41; or the sequence of SEQ ID NO: 42.

Various embodiments of amplification oligomers, including with respectto their sequences, are disclosed in the summary above, any of which canbe combined to the extent feasible with the features discussed above inthis section. In some embodiments, at least one probe oligomer isprovided. Some embodiments of detection probes that hybridize tocomplementary amplified sequences may be DNA or RNA oligomers, oroligomers that contain a combination of DNA and RNA nucleotides, oroligomers synthesized with a modified backbone, e.g., an oligomer thatincludes one or more 2′-methoxy substituted ribonucleotides. Probes usedfor detection of the amplified C1orf43 sequences may be unlabeled anddetected indirectly (e.g., by binding of another binding partner to amoiety on the probe) or may be labeled with a variety of detectablelabels. A detection probe oligomer may contain a 2′-methoxy backbone atone or more linkages in the nucleic acid backbone.

In some embodiments, a detection probe oligomer in accordance with thepresent disclosure further includes a label. Particularly suitablelabels include compounds that emit a detectable light signal, e.g.,fluorophores or luminescent (e.g., chemiluminescent) compounds that canbe detected in a homogeneous mixture. More than one label, and more thanone type of label, may be present on a particular probe, or detectionmay rely on using a mixture of probes in which each probe is labeledwith a compound that produces a detectable signal (see. e.g., U.S. Pat.Nos. 6,180,340 and 6,350,579, each incorporated by reference herein).Labels may be attached to a probe by various means including covalentlinkages, chelation, and ionic interactions, but in some embodiments thelabel is covalently attached. For example, in some embodiments, adetection probe has an attached chemiluminescent label such as, e.g., anacridinium ester (AE) compound (see. e.g., U.S. Pat. Nos. 5,185,439;5,639,604; 5,585,481; and 5,656,744; each incorporated by referenceherein), which in typical variations is attached to the probe by anon-nucleotide linker (see. e.g., U.S. Pat. Nos. 5,585,481; 5,656,744;and 5,639,604, each incorporated by reference herein).

A detection probe oligomer in accordance with the present disclosure mayfurther include a non-target-hybridizing sequence. In some applications,probes exhibiting at least some degree of self-complementarity aredesirable to facilitate detection of probe:target duplexes in a testsample without first requiring the removal of unhybridized probe priorto detection. Specific embodiments of such detection probes include, forexample, probes that form conformations held by intramolecularhybridization, such as conformations generally referred to as hairpins.Particularly suitable hairpin probes include a “molecular torch” (see.e.g., U.S. Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945,each incorporated by reference herein) and a “molecular beacon” (see.e.g., Tyagi et al., supra; U.S. Pat. Nos. 5,118,801 and 5,312,728,supra).

In yet other embodiments, a detection probe is a linear oligomer thatdoes not substantially form conformations held by intramolecular bonds.In some embodiments, the linear detection probe comprises an AE asdiscussed above. Detection probes comprising an AE, including lineardetection probes, can function as flasher probes or glower probes indual kinetic assays (DKAs). See, e.g., U.S. Pat. No. 5,840,873, which isincorporated by reference herein, for a description of flasher andglower probes and dual kinetic assays.

By way of example of detection oligomers comprising anon-target-hybridizing sequence, structures referred to as “molecularbeacons” comprise nucleic acid molecules having a target complementarysequence, an affinity pair (or nucleic acid arms) holding the probe in aclosed conformation in the absence of a target nucleic acid sequence,and a label pair that interacts when the probe is in a closedconformation. Hybridization of the target nucleic acid and the targetcomplementary sequence separates the members of the affinity pair,thereby shifting the probe to an open conformation. The shift to theopen conformation is detectable due to reduced interaction of the labelpair, which may be, for example, a fluorophore and a quencher (e.g.,DABCYL and EDANS). Molecular beacons are fully described in U.S. Pat.No. 5,925,517, the disclosure of which is hereby incorporated byreference. Molecular beacons useful for detecting C1orf43 nucleic acidsequences may be created by appending to either end of one of the probe(e.g., target-hybridizing) sequences disclosed herein, a first nucleicacid arm comprising a fluorophore and a second nucleic acid armcomprising a quencher moiety. In this configuration, a C1orf43 specificprobe sequence disclosed herein serves as the target-complementary“loop” portion of the resulting molecular beacon, while theself-complementary “arms” of the probe represent the “stem” portion ofthe probe.

Another example of a self-complementary hybridization assay probe thatmay be used in conjunction with the disclosure is a structure commonlyreferred to as a “molecular torch” (sometimes referred to simply as atorch). These self-reporting probes are designed to include distinctregions of self-complementarity (coined “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., a —(CH2)₉— linker) and which hybridize to one another underpredetermined hybridization assay conditions. When exposed to anappropriate target or denaturing conditions, the two complementaryregions (which may be fully or partially complementary) of the moleculartorch melt, leaving the target binding domain available forhybridization to a target sequence when the predetermined hybridizationassay conditions are restored. Molecular torches are designed so thatthe target binding domain favors hybridization to the target sequenceover the target closing domain. The target binding domain and the targetclosing domain of a molecular torch include interacting labels (e.g.,fluorescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized as opposed to when themolecular torch is hybridized to a target nucleic acid, therebypermitting detection of probe:target duplexes in a test sample in thepresence of unhybridized probe having a viable label associatedtherewith. Molecular torches are fully described in U.S. Pat. No.6,361,945, the disclosure of which is hereby incorporated by reference.

Molecular torches and molecular beacons in some embodiments are labeledwith an interactive pair of detectable labels. Examples of detectablelabels that are members of an interactive pair of labels include thosethat interact with each other by FRET or non-FRET energy transfermechanisms. Fluorescence resonance energy transfer (FRET) involves theradiationless transmission of energy quanta from the site of absorptionto the site of its utilization in the molecule, or system of molecules,by resonance interaction between chromophores, over distancesconsiderably greater than interatomic distances, without conversion tothermal energy, and without the donor and acceptor coming into kineticcollision. The “donor” is the moiety that initially absorbs the energy,and the “acceptor” is the moiety to which the energy is subsequentlytransferred. In addition to FRET, there are at least three other“non-FRET” energy transfer processes by which excitation energy can betransferred from a donor to an acceptor molecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other. This is the case, for example, when amolecular beacon is maintained in the closed state by formation of astem duplex, and fluorescent emission from a fluorophore attached to onearm of the probe is quenched by a quencher moiety on the opposite arm.

Exemplary label moieties for the disclosed molecular torches andmolecular beacons include a fluorophore and a second moiety havingfluorescence quenching properties (i.e., a “quencher”). In thisembodiment, the characteristic signal is likely fluorescence of aparticular wavelength, but alternatively could be a visible lightsignal. When fluorescence is involved, changes in emission are in someembodiments due to FRET, or to radiative energy transfer or non-FRETmodes. When a molecular beacon having a pair of interactive labels inthe closed state is stimulated by an appropriate frequency of light, afluorescent signal is generated at a first level, which may be very low.When this same probe is in the open state and is stimulated by anappropriate frequency of light, the fluorophore and the quenchermoieties are sufficiently separated from each other that energy transferbetween them is substantially precluded. Under that condition, thequencher moiety is unable to quench the fluorescence from thefluorophore moiety. If the fluorophore is stimulated by light energy ofan appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties.

Examples of donor/acceptor label pairs that may be used in connectionwith the disclosure, making no attempt to distinguish FRET from non-FRETpairs, include fluorescein/tetramethylrhodamine, IAEDANS/fluororescein,EDANS/DABCYL, coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPYFL, fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL,eosine/DABCYL, erythrosine/DABCYL, tetramethylrhodamine/DABCYL, TexasRed/DABCYL, CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2 and fluorescein/QSY7 dye.Those having an ordinary level of skill in the art will understand thatwhen donor and acceptor dyes are different, energy transfer can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence. When the donor and acceptor speciesare the same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Exemplary fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Exemplary quencher moieties that can be used asanother member of a donor-acceptor pair include DABCYL and the BLACKHOLE QUENCHER moieties which are available from Biosearch Technologies,Inc., (Novato, Calif.).

Oligomers that are not intended to be extended by a nucleic acidpolymerase, e.g., probe oligomers and capture oligomers, can include ablocker group that replaces the 3′ OH to prevent enzyme-mediatedextension of the oligomer in an amplification reaction. For example,blocked amplification oligomers and/or detection probes present duringamplification in some embodiments do not have a functional 3′ OH andinstead include one or more blocking groups located at or near the 3′end. A blocking group near the 3′ end is in some embodiments within fiveresidues of the 3′ end and is sufficiently large to limit binding of apolymerase to the oligomer, and other embodiments contain a blockinggroup covalently attached to the 3′ terminus. Many different chemicalgroups may be used to block the 3′ end, e.g., alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

While oligonucleotide probes of different lengths and base compositionmay be used for detecting C1orf43 nucleic acids, some embodiments ofprobes in this disclosure are from 10 to 60 bases in length, or between14 and 50 bases in length, or between 15 and 30 bases in length. A probeoligomer can be provided that is configured to specifically hybridize tothe amplicon discussed above.

Exemplary target hybridizing sequences for C1orf43 detection oligomersare SEQ ID NOs: 15, 17, 19, 21, 23, 25, and 59-72. Exemplary moleculartorch sequences for detecting a C1orf43 amplicon are SEQ ID NOs: 16, 18,and 20 (e.g., including the features noted in the sequence tableincluding a linker and labels). Exemplary molecular beacon sequences fordetecting a C1orf43 amplicon are SEQ ID NOs: 22 and 24 (e.g., includingthe features noted in the sequence table including labels). Exemplarylinear detection probe sequences are SEQ ID NOs: 15 and 59-72. Suchlinear detection probe sequences are suitable for use in, e.g.,hybridization protection and/or dual kinetic assay formats. SEQ ID NO:69 is an exemplary sequence suitable for use as a glower probe in a DKA.SEQ ID NOs: 15, 59, 68, and 70 are exemplary sequences suitable for useas a flasher probe in a DKA.

Various embodiments of a probe oligomer, including with respect to itssequence, are disclosed in the summary above, any of which can becombined to the extent feasible with the features discussed above inthis section.

In some embodiments, at least one capture oligomer is provided, e.g.,two, three, or four capture oligomers. It is understood that when two ormore capture oligomers are present, their target-hybridizing sequencesare different from each other. The one or more capture oligomerscomprise a target-hybridizing sequence configured to specificallyhybridize to C1orf43 nucleic acid, e.g., from 10 to 60 bases in length,or between 14 and 50 bases in length, or between 15 and 30 bases inlength. For example, in specific embodiments, at least one capture probehas the target-hybridizing sequence of SEQ ID NO: 5, 7, 9, 11, or 13.The target-hybridizing sequence is covalently attached to a sequence ormoiety that binds to an immobilized probe, e.g., an oligomer attached toa solid substrate, such as a bead.

In more specific embodiments, the capture oligomer includes a tailportion (e.g., a 3′ tail) that is not complementary to the C1orf43target sequence but that specifically hybridizes to a sequence of theimmobilized binding partner (e.g., immobilized probe), thereby servingas the moiety allowing the target nucleic acid to be separated fromother sample components, such as previously described in, e.g., U.S.Pat. No. 6,110,678, incorporated herein by reference. Any sequence maybe used in a tail region, which is can be 5 to 50 nt long, and certainembodiments include a substantially homopolymeric tail (“poly-Nsequence”) of at least 10 nt, e.g., 10 to 40 nt (e.g., A₁₀ to A₄₀), suchas 14 to 33 nt (e.g., A₁₄ to A₃₀ or T₃A₁₄ to T₃A₃₀), that bind to acomplementary immobilized sequence (e.g., poly-1) attached to a solidsupport, e.g., a matrix or particle. For example, in specificembodiments of a capture probe comprising a 3′ tail, at least onecapture probe has the sequence of SEQ ID NO: 4, 6, 8, 10, or 12.

Various embodiments of capture oligomers, including with respect to itssequence, are disclosed in the summary above, any of which can becombined to the extent feasible with the features discussed above inthis section.

Internal control oligomers can be provided, e.g., for confirming that anegative result is valid by establishing that conditions were suitablefor amplification. A control template that can be amplified by thecontrol amplification oligomers can also be provided. Control templatesmay be prepared according to known protocols. See, e.g., U.S. Pat. No.7,785,844, which is incorporated herein by reference, and whichdescribes an internal control consisting of an in vitro synthesizedtranscript containing a portion of HIV-1 sequence and a unique sequencetargeted by the internal control probe.

In certain aspects of the disclosure, a combination of at least twooligomers is provided for determining the presence or absence of aC1orf43 nucleic acid or quantifying a C1orf43 nucleic acid in a sample.In some embodiments, the C1orf43 nucleic acid is a human C1orf43 nucleicacid. In some embodiments, the C1orf43 nucleic acid is an mRNA, such asa human C1orf43 mRNA. In some embodiments, the oligomer combinationincludes at least two amplification oligomers suitable for amplifying atarget region of a C1orf43 target nucleic acid, e.g., having thesequence of SEQ ID NO: 1, 3, 39, 40, 41, or 42. In such embodiments, atleast one amplification oligomer comprises a target-hybridizing sequencein the sense orientation (“sense THS”) and at least one amplificationoligomer comprises a target-hybridizing sequence in the antisenseorientation (“antisense THS”), where the sense THS and antisense THS areeach configured to specifically hybridize to a target sequence within aC1orf43sequence. It is understood that the target-hybridizing sequencesare selected such that the sequence targeted by antisense THS issituated downstream of the sequence targeted by the sense THS (i.e., theat least two amplification oligomers are situated such that they flankthe target region to be amplified).

The oligomers can be provided in various combinations (e.g., kits orcompositions) as set forth in the summary above, e.g., comprising 2, 3,or 4 of a first amplification oligomer, second amplification oligomer,probe oligomer, capture oligomer, such as at least one firstamplification oligomer and at least one capture oligomer; a firstamplification oligomer and a second amplification oligomer, optionallyfurther comprising a probe oligomer; or at least one capture oligomer, afirst amplification oligomer, and a second amplification oligomer,optionally further comprising a probe oligomer.

In some embodiments, a combination of oligomers is provided as describedbelow in any of the examples or individual reactions described in theexamples.

Also provided by the disclosure is a reaction mixture for determiningthe presence or absence of a C1orf43 target nucleic acid or quantifyingthe amount thereof in a sample. A reaction mixture in accordance withthe present disclosure comprises at least one or more of the following:an oligomer combination as described herein for amplification of aC1orf43 target nucleic acid; a capture probe oligomer as describedherein for purifying the C1orf43 target nucleic acid; and a detectionprobe oligomer as described herein for determining the presence orabsence of a C1orf43 amplification product. In some embodiments, anyoligomer combination described above is present in the reaction mixture.The reaction mixture may further include a number of optional componentssuch as, for example, arrays of capture probe nucleic acids. For anamplification reaction mixture, the reaction mixture will typicallyinclude other reagents suitable for performing in vitro amplificationsuch as, e.g., buffers, salt solutions, appropriate nucleotidetriphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and/or ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase), and will typically include test sample components, in whicha C1orf43 target nucleic acid may or may not be present. In addition,for a reaction mixture that includes a detection probe together with anamplification oligomer combination, selection of amplification oligomersand detection probe oligomers for a reaction mixture are linked by acommon target region (i.e., the reaction mixture will include a probethat binds to a sequence amplifiable by an amplification oligomercombination of the reaction mixture).

Also provided by the subject disclosure are kits for practicing themethods as described herein. A kit in accordance with the presentdisclosure comprises at least one or more of the following: anamplification oligomer combination as described herein for amplificationof a C1orf43 target nucleic acid; at least one capture probe oligomer asdescribed herein for purifying the C1orf43 target nucleic acid; and atleast one detection probe oligomer as described herein for determiningthe presence or absence of a C1orf43 amplification product. In someembodiments, any oligomer combination described above is present in thekit. The kits may further include a number of optional components suchas, for example, arrays of capture probe nucleic acids. Other reagentsthat may be present in the kits include reagents suitable for performingin vitro amplification such as, e.g., buffers, salt solutions,appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP;and/or ATP, CTP, GTP and UTP), and/or enzymes (e.g., reversetranscriptase, and/or RNA polymerase). Oligomers as described herein maybe packaged in a variety of different embodiments, and those skilled inthe art will appreciate that the disclosure embraces many different kitconfigurations. In addition, for a kit that includes a detection probetogether with an amplification oligomer combination, selection ofamplification oligomers and detection probe oligomers for a kit arelinked by a common target region (i.e., the kit will include a probethat binds to a sequence amplifiable by an amplification oligomercombination of the kit). In certain embodiments, the kit furtherincludes a set of instructions for practicing methods in accordance withthe present disclosure, where the instructions may be associated with apackage insert and/or the packaging of the kit or the componentsthereof.

D. Methods and Uses

Any method disclosed herein is also to be understood as a disclosure ofcorresponding uses of materials involved in the method directed to thepurpose of the method. Any of the oligomers comprising C1orf43 sequenceand any combinations (e.g., kits and compositions) comprising such anoligomer are to be understood as also disclosed for use in detecting orquantifying C1orf43, and for use in the preparation of a composition fordetecting or quantifying C1orf43.

Broadly speaking, methods can comprise one or more of the followingcomponents: target capture, in which C1orf43 nucleic acid is annealed toa capture oligomer and optionally to an initial amplification oligomer;isolation, e.g., washing, to remove material not associated with acapture oligomer; linear amplification; exponential amplification; andamplicon detection, e.g., amplicon quantification, which may beperformed in real time with exponential amplification. Certainembodiments involve each of the foregoing steps. Certain embodimentsinvolve exponential amplification without linear amplification. Certainembodiments involve washing, isolation, and linear amplification.Certain embodiments involve exponential amplification and amplicondetection. Certain embodiments involve any two of the components listedabove. Certain embodiments involve any two components listed adjacentlyabove, e.g., washing and linear amplification, or linear amplificationand exponential amplification.

In some embodiments, amplification comprises (1) contacting the samplewith at least two oligomers for amplifying a C1orf43 nucleic acid targetregion corresponding to a C1orf43 target nucleic acid, where theoligomers include at least two amplification oligomers as describedabove (e.g., one or more oriented in the sense direction and one or moreoriented in the antisense direction for exponential amplification); (2)performing an in vitro nucleic acid amplification reaction, where anyC1orf43 target nucleic acid present in the sample is used as a templatefor generating an amplification product; and (3) detecting the presenceor absence of the amplification product, thereby determining thepresence or absence of C1orf43 in the sample, or quantifying the amountof C1orf43 nucleic acid in the sample.

In some embodiments, amplification comprises (1) contacting the samplewith at least four oligomers for amplifying a C1orf43 nucleic acidtarget region corresponding to a C1orf43 target nucleic acid, where theoligomers include at least two amplification oligomers for producing afirst amplicon as described above (e.g., one or more oriented in thesense direction and one or more oriented in the antisense direction forexponential amplification) and at least two amplification oligomers forproducing a second amplicon as described above (e.g., one or moreoriented in the sense direction and one or more oriented in theantisense direction for exponential amplification); (2) performing an invitro nucleic acid amplification reaction, where any C1orf43 targetnucleic acid present in the sample is used as a template for generatingan amplification product; and (3) detecting the presence or absence ofthe first or second amplicons, thereby determining the presence orabsence of C1orf43 in the sample, or quantifying the amount of C1orf43nucleic acid in the sample.

A detection method in accordance with the present disclosure can furtherinclude the step of obtaining the sample to be subjected to subsequentsteps of the method. In certain embodiments, “obtaining” a sample to beused includes, for example, receiving the sample at a testing facilityor other location where one or more steps of the method are performed,and/or retrieving the sample from a location (e.g., from storage orother depository) within a facility where one or more steps of themethod are performed.

In certain embodiments, the method further includes purifying theC1orf43 target nucleic acid from other components in the sample, e.g.,before an amplification, such as before a capture step. Suchpurification may include methods of separating and/or concentratingorganisms contained in a sample from other sample components, orremoving or degrading non-nucleic acid sample components, e.g., protein,carbohydrate, salt, lipid, etc. In some embodiments, DNA in the sampleis degraded, e.g., with DNase, and optionally removing or inactivatingthe DNase or removing degraded DNA.

In particular embodiments, purifying the target nucleic acid includescapturing the target nucleic acid to specifically or non-specificallyseparate the target nucleic acid from other sample components.Non-specific target capture methods may involve selective precipitationof nucleic acids from a substantially aqueous mixture, adherence ofnucleic acids to a support that is washed to remove other samplecomponents, or other means of physically separating nucleic acids from amixture that contains C1orf43 nucleic acid and other sample components.

Target capture typically occurs in a solution phase mixture thatcontains one or more capture probe oligomers that hybridize specificallyto the C1orf43 target sequence under hybridizing conditions, usually ata temperature higher than the Tm of thetail-sequence:immobilized-probe-sequence duplex. For embodimentscomprising a capture probe tail, the C1orf43-target:capture-probecomplex is captured by adjusting the hybridization conditions so thatthe capture probe tail hybridizes to the immobilized probe. Certainembodiments use a particulate solid support, such as paramagnetic beads.

Isolation can follow capture, wherein the complex on the solid supportis separated from other sample components. Isolation can be accomplishedby any appropriate technique, e.g., washing a support associated withthe C1orf43-target-sequence one or more times (e.g., 2 or 3 times) toremove other sample components and/or unbound oligomer. In embodimentsusing a particulate solid support, such as paramagnetic beads, particlesassociated with the C1orf43-target may be suspended in a washingsolution and retrieved from the washing solution, in some embodiments byusing magnetic attraction. To limit the number of handling steps, theC1orf43 target nucleic acid may be amplified by simply mixing theC1orf43 target sequence in the complex on the support with amplificationoligomers and proceeding with amplification steps.

Linear amplification can be performed, e.g., by contacting the targetnucleic acid sequence with a first phase amplification reaction mixturethat supports linear amplification of the target nucleic acid sequenceand lacks at least one component that is required for its exponentialamplification. In some embodiments, the first phase amplificationreaction mixture includes an amplification enzyme selected from areverse transcriptase, a polymerase, and a combination thereof. Thepolymerase is typically selected from an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, and acombination thereof. In some embodiments, the first phase amplificationreaction mixture further includes a ribonuclease (RNase), such as anRNase H or a reverse transcriptase with an RNase H activity. In someembodiments, the first phase amplification mixture includes a reversetranscriptase with an RNase H activity and an RNA polymerase.

In some embodiments, the first phase amplification mixture may alsoinclude an amplification oligonucleotide. The amplificationoligonucleotide can include a 5′ promoter sequence for an RNApolymerase, such as T7 RNA polymerase, and/or a blocked 3′ terminus thatprevents its enzymatic extension. In addition, the first phaseamplification mixture may sometimes include a blocker oligonucleotide toprevent enzymatic extension of the target nucleic sequence beyond adesired end-point.

As noted above, the key feature of the first phase amplificationreaction is its inability to support an exponential amplificationreaction because one or more components required for exponentialamplification are lacking, and/or an agent is present which inhibitsexponential amplification, and/or the temperature of the reactionmixture is not conducive to exponential amplification, etc. Withoutlimitation, the lacking component required for exponential amplificationand/or inhibitor and/or reaction condition may be selected from thefollowing group: an amplification oligonucleotide (e.g., anamplification oligonucleotide comprising a 5′ promoter sequence for anRNA polymerase, a non-promoter amplification oligonucleotide, or acombination thereof), an enzyme (e.g., a polymerase, such as an RNApolymerase), a nuclease (e.g., an exonuclease, an endonuclease, acleavase, an RNase, a phosphorylase, a glycosylase, etc), an enzymeco-factor, a chelator (e.g., EDTA or EGTA), ribonucleotide triphosphates(rNTPs), deoxyribonucleotide triphosphates (dNTPs), Mg²⁺, a salt, abuffer, an enzyme inhibitor, a blocking oligonucleotide, pH,temperature, salt concentration and a combination thereof. In somecases, the lacking component may be involved indirectly, such as anagent that reverses the effects of an inhibitor of exponentialamplification which is present in the first phase reaction.

Exponentially amplifying a C1orf43 target sequence utilizes an in vitroamplification reaction using at least two amplification oligomers thatflank a target region to be amplified.

In some embodiments, at least first and second amplification oligomersas described above are provided. In particular embodiments, the targetregion to be amplified corresponds to any amplicon discussed above.

Particularly suitable amplification oligomer combinations foramplification of these target regions are described above and in theexamples. In some embodiments, the target regions flanked by the firstand second amplification oligomers and by the third and fourthamplification oligomers are amplified in the same reaction mixture.Suitable amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification (TMA).

For example, some amplification methods that use TMA amplificationinclude the following steps. Briefly, the target nucleic acid thatcontains the sequence to be amplified is provided as single-strandednucleic acid (e.g., ssRNA such as C1orf43 mRNA) or is converted to asubstantially single-stranded state, e.g., by heat denaturation followedby rapid cooling. Those skilled in the art will appreciate that DNA canbe used in TMA; melting of double stranded nucleic acid (e.g., dsDNA)may be used to provide single-stranded target nucleic acids. A promoterprimer (e.g., a first amplification oligomer comprising a promoter asdescribed above) binds specifically to the target nucleic acid at itstarget sequence and a reverse transcriptase (RT) extends the 3′ end ofthe promoter primer using the target strand as a template to create acDNA extension product, resulting in an RNA:DNA duplex if ssRNA was theoriginal template. Thus, in some embodiments, a cDNA comprising thesequence of a C1orf43 amplicon disclosed herein is produced. An RNase(e.g., RNase H) digests the RNA strand of the RNA:DNA duplex and asecond primer binds specifically to its target sequence, which islocated on the cDNA strand downstream from the promoter primer end. RTsynthesizes a new DNA strand by extending the 3′ end of the other primerusing the first cDNA template to create a dsDNA that contains afunctional promoter sequence. An RNA polymerase specific for thepromoter sequence then initiates transcription to produce RNAtranscripts that are about 100 to 1000 amplified copies (“amplicons”) ofthe initial target strand in the reaction. Amplification continues whenthe other primer binds specifically to its target sequence in each ofthe amplicons and RT creates a DNA copy from the amplicon RNA templateto produce an RNA:DNA duplex. RNase in the reaction mixture digests theamplicon RNA from the RNA:DNA duplex and the promoter primer bindsspecifically to its complementary sequence in the newly synthesized DNA.RT extends the 3′ end of the promoter primer to create a dsDNA thatcontains a functional promoter to which the RNA polymerase binds totranscribe additional amplicons that are complementary to the targetstrand. The autocatalytic cycles of making more amplicon copies repeatduring the course of the reaction resulting in about a billion-foldamplification of the target nucleic acid present in the sample. Theamplified products may be detected in real-time during amplification, orat the end of the amplification reaction by using a probe that bindsspecifically to a target sequence contained in the amplified products.Detection of a signal resulting from the bound probes indicates thepresence of the target nucleic acid in the sample.

In some embodiments, the method utilizes a “reverse” TMA reaction. Insuch variations, the initial or “forward” amplification oligomer is apriming oligonucleotide that hybridizes to the target nucleic acid inthe vicinity of the 3′-end of the target region. A reverse transcriptase(RT) synthesizes a cDNA strand by extending the 3′-end of the primerusing the target nucleic acid as a template. The other or “reverse”amplification oligomer is a promoter primer or promoter provider havinga target-hybridizing sequence configured to hybridize to atarget-sequence contained within the synthesized cDNA strand. Where thesecond amplification oligomer is a promoter primer, RT extends the 3′end of the promoter primer using the cDNA strand as a template to createa second, cDNA copy of the target sequence strand, thereby creating adsDNA that contains a functional promoter sequence. Amplification thencontinues essentially as described above in the preceding paragraph forinitiation of transcription from the promoter sequence utilizing an RNApolymerase. Alternatively, where the second amplification oligomer is apromoter provider, a terminating oligonucleotide, which hybridizes to atarget sequence that is in the vicinity to the 5′-end of the targetregion, is typically utilized to terminate extension of the primingoligomer at the 3′-end of the terminating oligonucleotide, therebyproviding a defined 3′-end for the initial cDNA strand synthesized byextension from the priming oligomer. The target-hybridizing sequence ofthe promoter provider then hybridizes to the defined 3′-end of theinitial cDNA strand, and the 3′-end of the cDNA strand is extended toadd sequence complementary to the promoter sequence of the promoterprovider, resulting in the formation of a double-stranded promotersequence. The initial cDNA strand is then used a template to transcribemultiple RNA transcripts complementary to the initial cDNA strand, notincluding the promoter portion, using an RNA polymerase that recognizesthe double-stranded promoter and initiates transcription therefrom. Eachof these RNA transcripts is then available to serve as a template forfurther amplification from the first priming amplification oligomer.

The detection step may be performed using any of a variety of knowntechniques to detect a signal specifically associated with the targetsequence, such as, e.g., by hybridizing the target C1orf43 nucleic acid(e.g., a C1orf43 amplification product) with a labeled detection probeand detecting a signal resulting from the labeled probe. The detectionstep may also provide additional information on the target sequence,such as, e.g., all or a portion of its nucleic acid base sequence.Detection may be performed after an amplification reaction is completed,or may be performed simultaneously with amplifying the target region,e.g., in real time. Alternatively, detection may be performed on nucleicacid released from cells in a sample, e.g., C1orf43 RNA released bylysing such cells. In one embodiment, the detection step allowshomogeneous detection, e.g., detection of the hybridized probe withoutremoval of unhybridized probe from the mixture (see, e.g., U.S. Pat.Nos. 5,639,604 and 5,283,174, each incorporated by reference herein). Insome embodiments, the nucleic acids are associated with a surface thatresults in a physical change, such as a detectable electrical change.Amplified nucleic acids may be detected by concentrating them in or on amatrix and detecting the nucleic acids or dyes associated with them(e.g., an intercalating agent such as ethidium bromide or cyber green),or detecting an increase in dye associated with nucleic acid in solutionphase. Other methods of detection may use nucleic acid detection probesthat are configured to specifically hybridize to a sequence in theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481; each incorporated by referenceherein). Directly or indirectly labeled probes that specificallyassociate with the amplified product provide a detectable signal thatindicates the presence of the target nucleic acid in the sample. Inparticular, the amplified product will contain a target sequence in orcomplementary to a sequence in the C1orf43 nucleic acid, and a probewill bind directly or indirectly to a sequence contained in theamplified product to indicate the presence of C1orf43 nucleic acid inthe tested sample.

In embodiments that detect the amplified product near or at the end ofthe amplification step, a linear detection probe may be used to providea signal to indicate hybridization of the probe to the amplifiedproduct. Such detection may use a hybridization protection assay format.For example, such detection may use a luminescently labeled probe thathybridizes to target nucleic acid. Luminescent label is then hydrolyzedfrom non-hybridized probe. Detection is performed by chemiluminescenceusing a luminometer (see, e.g., International Patent Application Pub.No. WO 89/002476, incorporated by reference herein). Detection can bemultiplexed with detection of an additional target, e.g., using a dualkinetic assay format, in which one of the C1orf43 target and theadditional target is detected with a flasher probe (showing relativelyfast kinetics (for example, with signal declining substantially from thepeak level (e.g., 10-fold) at or before 25 milliseconds followinginitiation of signal, such as the triggering of a chemiluminescentreaction) and the other of the C1orf43 target and the additional targetis detected with a glower probe (showing relatively slow kinetics, forexample, with signal continuing at or above 15% of the peak value as of20 or 25 milliseconds after the peak value occurred and/ordistinguishably later than signal from the flasher probe). In otherembodiments that use real-time detection, the detection probe may be ahairpin probe such as, for example, a molecular beacon, molecular torch,or hybridization switch probe that is labeled with a reporter moietythat is detected when the probe binds to amplified product. Such probesmay comprise target-hybridizing sequences and non-target-hybridizingsequences. Various forms of such probes have been described previously(see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 5,925,517; 6,150,097;6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US PatentApplication Pub. Nos. 20060068417A1 and 20060194240A1; each incorporatedby reference herein).

In some embodiments, a molecular torch (sometimes referred to simply asa torch) is used for detection. In some embodiments, the torch is aprobe oligomer as disclosed above.

In general, methods involving quantification can involve the step ofconsulting a standard curve (which can be, e.g., in the form of anequation or graph, including digital representations thereof) thatrelates pre-amplification amounts of analyte polynucleotide andpost-amplification amounts of analyte amplicon.

Since real-time amplification reactions advantageously featurequantitative relationships between the number of analyte polynucleotidesinput into the reaction and the number of analyte amplicons synthesizedas a function of time, the number of analyte polynucleotides present ina test sample can be determined using a standard curve. For example, aplurality of amplification reactions containing known amounts of apolynucleotide standard can be run in parallel with an amplificationreaction prepared using a test sample containing an unknown number ofanalyte polynucleotides. Alternatively, a standard curve can be preparedin advance so that it is unnecessary to prepare a curve each time ananalytical procedure is carried out. Such a curve prepared in advancecan even be stored electronically in a memory device of a testinginstrument. A standard curve having pre-amplification amounts of thepolynucleotide standard on a first axis and some indicia of the timerequired to effect a certain level of nucleic acid amplification (suchas a time-of-emergence above a background signal) on a second axis isthen prepared. The post-amplification amount of analyte ampliconmeasured for the test reaction is then located on the post-amplificationaxis of the standard curve. The corresponding value on the other axis ofthe curve represents the pre-amplification amount of analytepolynucleotide that was present in the test reaction. Thus, determiningthe number of molecules of analyte polynucleotide present in the testsample is accomplished by consulting the standard curve, or moreparticularly by comparing the quantitative results obtained for the testsample with the standard curve, a procedure that will be familiar tothose having an ordinary level of skill in the art.

The procedures described herein can be used to quantify analytepolynucleotides (e.g., C1orf43 nucleic acid) present in a test sample.Indeed, if a plurality of standard control amplification reactions areinitiated using known numbers of an analyte polynucleotide standard, andif a test reaction that includes an unknown number of analytepolynucleotide molecules is carried out, then it becomes possible aftermeasuring the time required to effect a certain level of amplificationin each reaction to determine the number of analyte polynucleotidemolecules that must have been present in the test sample. Therelationship between the number of analyte polynucleotide moleculesinput into standard amplification reaction and the time required toeffect a certain level of amplification is conveniently establishedusing a graph or an equation corresponding to the graph. Determining thenumber of analyte polynucleotide molecules present in a test sample issimply a matter of determining from the standard graph or equation thenumber of analyte polynucleotide molecules that correspond to a measuredanalyte amplicon signal strength. This illustrates how analytepolynucleotide standards can be used in connection with polynucleotideamplification reactions to quantify pre-amplification amounts of analytepolynucleotide contained in test samples.

Levels can be expressed in various ways, e.g., as concentrations,absolute numbers of copies, mass, emergence time, or RLU or RFU. Levelscan be logarithmic or arithmetic. Levels can be converted betweendifferent forms of expression. For example, RFU versus time can beconverted to an emergence time, and emergence time can be converted to alogarithmic value using a calibration curve. As a further example, thelogarithmic value can be converted to an arithmetic value. In someembodiments, a calibration curve or other appropriate standard is usedto aid in comparing a level to a predetermined threshold.

In some embodiments, at least one nucleic acid of a microbe or pathogenis detected in addition to a C1orf43 nucleic acid. As shown in theexamples, the disclosure provides compositions and methods that do notcross-react with a wide variety of microbes including many pathogens.Accordingly, compositions and methods provided herein are suitable forcombination with known methods and compositions for detecting nucleicacids of microbes or pathogens. Exemplary microbes and pathogens includethose mentioned below in Example 7. In some embodiments, the nucleicacid of a microbe or pathogen is a human papillomavirus nucleic acid,Chlamydia trachomatis nucleic acid, Neisseria gonorrheae nucleic acid,Trichomonas vaginalis nucleic acid, or Mycoplasma genitalium nucleicacid. In some embodiments, the nucleic acid of a microbe or pathogen isa nucleic acid detectable according to a method provided in any one ofthe Aptima Combo 2® Assay (Hologic; Document No. 205446 Rev. 003, March2017); The Aptima® Trichomonas vaginalis Assay (Hologic; Document no.503684 Rev. 002, July 2017); or the Aptima Mycoplasma genitalium Assay(Hologic; Document No. AW-14170-001 Rev. 005, May 2017), each of whichare available from Hologic, e.g., via the Hologic website, and which areincorporated herein by reference.

In some embodiments, at least one RNA other than C1orf43 is detected.The cross-reactivity data provided below indicates that compositions andmethods according to this disclosure can be highly specific.Accordingly, compositions and methods provided herein are suitable forcombination with known methods and compositions for detecting otherRNAs, such as other human mRNAs. Exemplary RNAs other than C1orf43include any RNA suitable for use as an internal control, e.g., forspecimen processing and amplification, such as RNA from BacteriophageMS2; and any RNA proposed for use in calibration in Eisenberg et al.,Trends in Genetics 29:569-574 (2013) (which is incorporated herein byreference), such as human mRNAs CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A,REEP5, SNRPD3, VCP, and VPS29; additional information and furtherdiscussion can be found in Eisenberg et al.

Detection of C1orf43 can be used to validate negative results in methodscomprising detection of other nucleic acids by indicating that thesample contained C1orf43-expressing cells, that the steps of the method(e.g., some or all of sample processing, target capture, amplification,and probe detection) were performed properly, and that the reagents used(e.g., enzymes and nucleoside triphosphates) were not compromised.

EXAMPLES

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

General Reagents and Methods. Target capture was performed with acapture oligomer (0.05 pmol/μl) and promoter primer (0.02 pmol/μl) witha sample in a volume of 500 μl. Incubation, isolation, and wash stepswere essentially as described in Hologic, Inc., The Aptima® Trichomonasvaginalis Assay, Document no. 503684 Rev. 002, July 2017, available onthe Hologic website, which is incorporated herein by reference.

Unless otherwise indicated, amplifications were performed isothermallyusing biphasic transcription-mediated amplification (TMA) with T7 RNApolymerase and reverse transcriptase, in which last primer added in thebiphasic procedures was the nonT7 primer. Biphasic TMA was carried outessentially as described in U.S. Pat. No. 9,139,870, which isincorporated herein by reference. During the exponential amplificationphase, the promoter primer concentration was 0.05 pmol/μl and the nonT7and probe oligomer concentrations were each 0.15 pmol/μl.

Detection used molecular torches as probe oligomers which contained a5′-fluorophore (e.g., FAM or ROX) and a 3′-quencher (e.g., DABCYL)(“5F3D” for FAM and DABCYL or “5R3D” for ROX and DABCYL). Torches arediscussed in detail in U.S. Pat. No. 6,849,412, which is incorporated byreference. Torches generally contained a —(CH₂)₉-linker near the 3′-end(e.g., between the 5^(th) and 6^(th) or between the 4^(th) and 5^(th)nucleotides from the 3′-end). Target capture was performed essentiallyas described in U.S. Pat. No. 8,034,554, which is incorporated herein byreference.

Separate internal control oligomers and template were used in someexperiments to verify reagent presence and activity, etc., and are notspecifically discussed below. Exemplary internal control oligomers andtemplate are discussed in U.S. Pat. No. 7,785,844, which is incorporatedherein by reference.

Example 1—Amplification and Detection Oligomer Screening

A series of amplification and probe oligomers were designed to evaluateamplification and detection of C1orf43 nucleic acid. Experiments wereperformed with the following combinations of oligomers.

TABLE 1 Exemplary oligomer combinations. Promoter- nonT7 Probe primerprimer oligomer Condition sequence sequence sequence 1 SEQ ID NO: 32 SEQID NO: 35 SEQ ID NO: 14 2 SEQ ID NO: 26 SEQ ID NO: 34 SEQ ID NO: 14 3SEQ ID NO: 30 SEQ ID NO: 37 SEQ ID NO: 14 4 SEQ ID NO: 28 SEQ ID NO: 36SEQ ID NO: 14 5 SEQ ID NO: 32 SEQ ID NO: 36 SEQ ID NO: 14 6 SEQ ID NO:28 SEQ ID NO: 38 SEQ ID NO: 14 1A SEQ ID NO: 32 SEQ ID NO: 35 SEQ ID NO:21 2A SEQ ID NO: 26 SEQ ID NO: 34 SEQ ID NO: 21 3A SEQ ID NO: 30 SEQ IDNO: 37 SEQ ID NO: 21 4A SEQ ID NO: 28 SEQ ID NO: 36 SEQ ID NO: 21 5A SEQID NO: 32 SEQ ID NO: 36 SEQ ID NO: 21 6A SEQ ID NO: 28 SEQ ID NO: 38 SEQID NO: 21 1B SEQ ID NO: 32 SEQ ID NO: 35 SEQ ID NO: 16 2B SEQ ID NO: 26SEQ ID NO: 34 SEQ ID NO: 16 3B SEQ ID NO: 30 SEQ ID NO: 37 SEQ ID NO: 164B SEQ ID NO: 28 SEQ ID NO: 36 SEQ ID NO: 16 5B SEQ ID NO: 32 SEQ ID NO:36 SEQ ID NO: 16 6B SEQ ID NO: 28 SEQ ID NO: 38 SEQ ID NO: 16

An in vitro transcript (IVT) of C1orf43 was prepared from a DNA havingthe sequence of SEQ ID NO: 3. Samples were prepared containing the IVTat either 10⁴ or 10⁶ copies (cp) per reaction, along with a negativecontrol lacking the IVT and used directly for amplification, without acapture step.

Amplification and detection results obtained with the exemplary oligomercombinations shown above are given in terms of TTime below (average ofduplicates). Negative control results (single replicate except forcondition 1 (2 replicates)) were obtained for conditions 1-6 and 2A-6Aand were as expected (no false positives; not shown).

TABLE 2 Amplification and detection results with exemplary oligomercombinations. TTime (min), TTime (min), Condition 10⁴ cp IVT 10⁶ cp IVT1 17.67 12.14 2 16.82 12.62 3 16.28 12.08 4 15.64 11.55 5 23.24 13.72 618.09 13.64 1A 17.50 11.75 2A 17.04 13.05 3A 16.07 11.97 4A 15.40 11.425A 21.12 13.60 6A 17.41 13.52 1B 12.60 9.16 2B 13.85 9.71 3B 14.96 9.024B 15.51 10.12 5B 16.68 11.10 6B 12.85 12.01

Example 2—Capture Oligomer Screening

Samples were prepared containing 10², 10³, 10⁴, or 10⁶ copies perreaction of the IVT described above in Example 1. Target capture wasperformed in separate experiments using capture oligomers with thefollowing sequences: SEQ ID NO: 4, 10, 8, and 6, followed byamplification and detection of the captured material with an exemplaryoligomer combination described above. Results (averages of triplicates)are shown below. Negative control (no IVT) results were obtained foreach capture oligomer and were as expected (no false positives; notshown).

TABLE 3 Results with exemplary capture oligomers. Capture oligomer TTime(min) SEQ ID NO 10² cp IVT 10³ cp IVT 10⁴ cp IVT 10⁶ cp IVT 4 18.1116.36 14.08 10.48 10 15.33 14.71 12.82 10.08 8 16.84 15.71 13.77 10.34 617.89 16.16 14.16 10.50

These results also show that the capture, amplification, and detectionof C1orf43 is useful for quantification in that TTime could be linearlyregressed against the logarithm of the number of copies to provide acalibration curve (not shown). The calibration curve slope in thedifferent experiments was between about −1.2 and −1.7 log₁₀ copies perminute of TTime.

Example 3—Tissue Type Screening

To confirm the utility of C1orf43 capture, amplification, and detectionwith samples from various tissues, an exemplary capture oligomer and anexemplary oligomer combination described above were used to capture,amplify, and detect C1orf43 mRNA from total RNA samples from the humantissues listed below. Total RNA samples were obtained from Biochain. Allsamples were run as a single replicate (1 ng of total RNA per reaction)except for penis (duplicate) and a negative control (triplicate) whichgave results as expected (no false positives; not shown). Results, shownas TTime values, were consistent with approximately 10,000 copies ofC1orf43 mRNA per sample or more based on calibration data (not shown).

TABLE 4 Results with different tissues. TTime Tissue type (min) Bladder11.48 Ductus deferens 10.65 Epididymis 10.66 Kidney 10.71 Lymph node12.96 Pancreas 13.13 Peripheral 13.19 blood lymphocyte Penis 11.66Prostate 1 12.45 Seminal vesicle 1 11.13 Spleen 12.45 Seminal vesicle 212.74 Prostate 2 10.59

Example 4—Performance with Clinical Vaginal Swab Samples

To confirm the utility of C1orf43 capture, amplification, and detectionwith clinical samples, an exemplary capture oligomer and an exemplaryoligomer combination described above were used to capture, amplify, anddetect C1orf43 mRNA from clinical vaginal swab samples.

Vaginal swabs were collected from patients per instructions in theAptima Unisex Swab Collection Kit for Endocervical and Male UrethralSwab Specimens (Hologic) where the specimen collection swabs are placedimmediately in the transport tube which contains a stabilization buffer,the swab shaft is broken off at the score line and the tube isre-capped. The input material from the vaginal swab specimen transporttube is 400 ul of the solution pipetted directly into the assay tube.

All samples were run as a single replicate except for sample 5(duplicate). Results, shown as TTime values, were consistent withapproximately 70,000 copies of C1orf43 mRNA per sample or more forsamples 1-13 based on calibration data (not shown).

TABLE 5 Results with clinical vaginal swab samples. Vaginal swab TTimesample (min) 1 11.12 2 10.1 3 8.41 4 8.95 5 8.76 6 8.39 7 9.03 8 9.05 98.92 10 8.93 11 10.41 12 11.06 13 9.67

Example 5—Testing on Processed Urine Samples

To confirm utility with an alternative clinical sample type, fifteenhuman processed post-DRE urine samples were tested. The urine wascollected from patients and processed using the PROGENSA PCA3 UrineSpecimen Transport Kit (Gen-Probe/Hologic) where the whole urine sampleis added to the urine specimen transport tube which contains PCA3 urinetransport medium. The processed urine input for the assay is 400 ul fromthe specimen tube per assay pipetted into the assay tube. An exemplarycapture oligomer and an exemplary oligomer combination described abovewere used to capture, amplify, and detect C1orf43 mRNA from theprocessed urine samples.

All samples were run as duplicates, including a negative control (notshown; no false positives). Results, shown as TTime values, wereconsistent with approximately 50,000 copies of C1orf43 mRNA per sampleor more for all tested samples based on calibration data (not shown).

TABLE 6 Results with processed urine samples. Whole urine TTime sample(min) 14 15.99 15 15.44 16 15.85 17 15.48 18 16.04 19 15.34 20 14.50 2115.94 22 14.93 23 13.92 24 14.10 25 14.50 26 14.23 27 13.88 28 14.92

Example 6—Testing on ThinPrep® Samples

To confirm utility with a further alternative clinical sample type, 197ThinPrep® clinical samples were tested. ThinPrep® clinical samples arecervical samples processed by immersion in a preservative solution withcirculation to remove debris, mucus, and other acellular materialfollowed by cell collection from the solution via filtration for furtheranalysis. The ThinPrep Pap test was used to collect the cervical sample.The ThinPrep liquid cytology specimen vial contains PreservCyt solutionwhich is then prepared for processing with the assay using the AptimaSpecimen Transfer Kit where 1 ml of ThinPrep liquid cytology specimen istransferred to the Aptima Specimen Transfer tube which contains ˜3 mlsample transport media. The processed ThinPrep sample input for theassay is 400 ul from the specimen tube per assay pipetted into the assaytube.

An exemplary capture oligomer and an exemplary oligomer combinationdescribed above were used to capture, amplify, and detect C1orf43 mRNAfrom the processed ThinPrep® samples.

In all samples, C1orf43 was detected; the low was 22 cp/mL and the highwas 263,857 cp/mL. In 75% of the clinical samples the total cps/mL wasunder 25,000 cp/mL, with the majority being less than 5,000 cp/mL. Meancp/mL was 22,290 cp/mL. All samples were run as duplicates, including anegative control (not shown; no false positives). Reproducibility wascharacterized by running 12 of the samples again on a different day witha different reagent batch. The geometric mean observed difference fromthe first measurement over these 12 samples was 0.15 log₁₀ cp/ml and therange was 0.04-0.32 log₁₀ cp/ml.

Eight negative samples were prepared, starting from preservativesolution without cervical material, and no false positive C1orf43results were observed.

Example 7—Testing of Specificity and Cross-Reactivity

To confirm the specificity of C1orf43 capture, amplification, anddetection, the procedure was performed with a series of pools ofmicrobes at high titer (4.4×10⁴ IFU/ml for C. trachomatis; 4.6×10⁴cells/ml for T. vaginalis; 10⁴ TCID₅₀/ml for herpes simplex viruses(HSV); 10⁶ copies/ml for HIV; 10⁶ colony forming units/ml for allothers). The microbes in each pool were as follows:

-   -   Acinetobacter iwoffii, Alcaligenes faecalis, Atopobium vaginae,        and Bacteroides fragilis    -   Campylobacter jejuni, Candida krusei, Candida lusitaniae, and        Chlamydia trachomatis    -   Corynebacterium genitalium, Cryptococcus neoformans, Eggerthella        lenta, and Enterobacter cloacae    -   Enterococcus faecalis, Escherichia coli, Fusobacterium        nucleatum, and Haemophilus ducreyi    -   Klebsiella pneumoniae, Lactobacillus acidophilus, and        Lactobacillus iners    -   Lactobacillus mucosae, Leptotrichia bucalis, Listeria        monocytogenes, and Megasphera elsdenii    -   Mobiluncos cutrisii, Neisseria gonorrheae, Peptostreptococcus        magnus, and Prevotella bivia    -   Propionibacterium acnes, Proteus vulgaris, Pseudomonas        aeruginosa, and Staphylococcus aureus    -   Staphylococcus epidermidis, Streptococcus agalactiae,        Streptococcus pyogenes, and Trichomonas vaginalis    -   Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma        hominis    -   Herpes simplex virus I, Herpes simplex virus II, and HIV    -   Actinomyces israelii    -   Candida dubliensis, Candida albicans, Candida glabrata, Candida        parasilopsis, and Candida tropicalis    -   Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus        gasseri, and Lactobacillus jensenii

Testing was negative for C1orf43 for all pools except the poolcontaining HSV and HIV. HSV and HIV were grown using a human cell lineand thus were thought to contain remnants of human nucleic acidincluding C1orf43.

Example 8—Testing with HPA Detection in Uniplex and Duplex Using an HPVTarget Nucleic Acid

A number of labeled linear detection probes were synthesized and labeledwith an acridinium ester (AE) detectable label for use with ahybridization protection assay (HPA) format. In this example, the lineardetection probes were labeled with an AE having fast kinetics, thusconfiguring the labeled detection probe to function as a flasher probein a dual kinetic assay (DKA) (see, e.g., U.S. Pat. No. 5,840,873 for adescription of flasher/glower probes used with dual kinetic assays).Detection probes comprising the sequences of SEQ ID NOs:15, 59, 68, 69,70, or 71 were synthesized and labeled with a fast kinetics AE. Each ofthe six different detection probes were individually added to a reactionwell containing 1000 copies per mL of a C1orf43 in vitro transcript.These reaction wells were then placed on a luminometer and the relativelight units (RLU) for each AE labeled detection probe was measured over50 milliseconds. The measured RLU data is presented in Table 7.

TABLE 7 Flasher Probe Kinetics. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID Milliseconds NO: 15 NO: 71 NO: 59 NO: 70 NO: 69 NO: 68 1 — 0 3 — — 12 745 1,532 997 548 694 461 3 6,989 10,058 7,163 4,897 5,053 4,789 417,973 20,067 15,707 11,659 10,312 12,084 5 24,571 24,586 20,169 15,97812,035 16,646 6 23,654 24,721 19,449 18,047 11,701 18,161 7 20,48521,325 18,078 18,186 11,354 17,975 8 17,339 18,102 15,877 16,957 10,62117,093 9 14,324 14,474 13,575 15,371 9,734 15,508 10 12,045 11,41511,547 13,651 8,808 13,675 11 10,114 9,180 9,588 11,954 7,818 11,892 128,371 7,462 8,012 10,542 6,751 10,283 13 6,887 6,132 6,717 9,026 6,0958,844 14 5,598 4,999 5,591 7,641 5,336 7,566 15 4,506 4,068 4,796 6,2714,835 6,397 16 3,744 3,269 4,116 5,271 4,230 5,366 17 3,054 2,669 3,4314,408 4,218 4,466 18 2,550 2,192 3,050 3,674 3,973 3,809 19 2,106 1,8012,563 3,164 3,523 3,271 20 1,765 1,496 2,267 2,669 3,446 2,735 21 1,4801,229 2,006 2,245 3,137 2,320 22 1,227 1,040 1,758 1,942 2,957 1,958 231,043 877 1,494 1,639 2,970 1,676 24 899 755 1,406 1,375 2,867 1,431 25781 643 1,245 1,206 2,867 1,223 26 684 547 1,186 1,085 2,790 1,060 27618 471 1,091 930 2,572 927 28 540 422 1,019 814 2,520 816 29 482 3711,003 745 2,417 710 30 438 333 915 679 2,417 648 31 400 303 890 6022,507 564 32 367 284 852 548 2,276 509 33 348 258 846 517 2,379 455 34330 238 799 497 2,199 428 35 299 223 802 464 2,122 394 36 283 213 742423 2,186 363 37 270 198 755 397 2,276 341 38 250 190 723 380 2,160 31339 243 182 733 362 2,109 302 40 239 175 698 342 1,993 286 41 221 164 673317 2,122 263 42 213 167 635 317 2,070 255 43 204 160 657 306 1,890 25344 198 157 664 296 2,057 226 45 193 146 632 289 2,032 218 46 196 145 629289 2,006 225 47 189 142 616 279 1,774 219 48 184 143 623 275 2,057 20649 182 135 619 269 1,800 199 50 176 139 591 260 1,929 188

For DKA, it is preferred that the detection probes labeled with theflasher AE has a fast kinetics so that the flasher signal can bedifferentiated from the glower signal. The above data show that alllabeled detection probes except the labeled detection probe comprisingSEQ ID NO:69 have good flasher kinetics because by 25 milliseconds, allRLU values had fallen below 1,300 RLU. Labeled detection probescomprising SEQ ID NOs:15 & 71 showed the best kinetics in this example,having RLU values below 800 at the 25 millisecond time point. These twolabeled detection probes were selected for use in a multiplexed assayfor the detection of human papilloma virus nucleic acids. It is notablefrom the above results, that all six labeled detection probes would beuseful for a non-DKA format, and further that the labeled detectionprobe comprising SEQ ID NO:69 may be useful as a glower detection probein a DKA.

Cellular Controls Using Linear Probes and DKA: A multiplex assay wasperformed for detecting human papilloma virus and a cellular control ina single reaction well. The assay was performed in DKA format, with alinear detection probe for detecting the cellular control beingdetectably labeled as a flasher (faster RLU kinetics) and a number oflinear detection probes for the detection of various HPV types beinglabeled as a glower (slower RLU kinetics). For amplifying and detectingC1orf43 from the cellular control, the reaction used one of thedetection probes comprising SEQ ID NOs:15 & 71 and labeled with AEmoieties, along with a promoter primer comprising the sequence of SEQ IDNO:30 and a non-T7 primer comprising the sequence of SEQ ID NO:34. Foramplification and detection of the HPV target nucleic acid, the reactionused the APTIMA HPV Assay (Hologic, Inc., San Diego, Calif.). Theinternal control was removed from this assay kit.

Samples for this example included SiHa cells and C33A cells (bothavailable from ATCC, Manassas, Va.). SiHa cells comprise both C1orf43nucleic acids and HPV nucleic acids. C33A cells comprise C1orf43 nucleicacids, but no HPV nucleic acids. The sample conditions were set-up asfollows: (i) 1,000 C33A cells per mL, (ii) 10 SiHa cells per mL, (iii)25 SiHa cells per mL, (iv) 250 SiHa cells per mL, (v) 10 SiHa cells and100,000 C33A cells per mL, (vi) 25 SiHa cells and 100,000 C33A cells permL, (vii) 500 SiHa cells and 1,000 C33A cells per mL, and (viii) 250SiHa cells and 1,000 C33A cells per mL. Condition (i) illustratesamplification and detection of the C1orf43 target nucleic acid.Conditions (ii) to (iv) illustrate amplification and detection of boththe C1orf43 target nucleic acid and the HPV nucleic acid. Conditions (v)to (viii) illustrate the amplification and detection of both the C1orf43target nucleic acid and the HPV nucleic acid in an environmentcontaining a high number of HPV negative cells relative to the number ofHPV positive cells. These conditions (v) to (viii) are more similar to acervical cell swab sample wherein not all collected cells are HPVpositive cells. Assays were set-up so that each of SEQ ID NOs:15 & 71were tested in duplicate against all 8 sample conditions. Multiplexassays were performed as is generally described in the APTIMA HPA Assayinstructions for use. Results are shown in Table 8 as RLU data.

TABLE 8 HPV & C1orf43 DKA results. DKA. DKA. C1** Assay AHPV Assay. AHPVAssay. AHPV* only. C1 assay C1 assay Sample Assay SEQ ID NOs: SEQ IDNOs: SEQ ID NOs: condition only. 15, 30 & 34. 15, 30 & 34. 30, 34 & 71.(i): 17,148 224,242 745,073 256,117 C33A 1,000 cells/mL (ii): 984,509208,512 922,587 353,563 10 SiHa cells/mL (iii): 915,145 315,0781,658,001 684,996 25 SiHa cells/mL (iv): 1,068,970 190,418 1,753,4841,228,866 250 SiHa cells/mL (v): 64,768 237,514 691,743 303,028 10 SiHacells/mL with 100,000 C33A cells/mL (vi): 83,018 248,521 717,803 355,70025 SiHa cells/mL with 100,000 C33A cells/mL (vii): 1,076,716 247,8091,766,458 1,308,922 500 SiHa cells/mL with 1,000 C33A cells/mL (viii):973,889 293,979 1,337,878 676,534 250 SiHa cells/mL with 1,000 C33Acells/mL *APTIMA HPV Assay (AHPV Assay). **C1 Assay (Cellularity Controlassay using a labeled detection probe oligomer comprising a sequence ofSEQ ID NO: 15 or 71).

These data in Table 8 illustrate a robust and consistent flasher signalfor the detection of C1orf43 target nucleic acid using the C1amplification and detection oligomers comprising sequences of SEQ IDNOs:15, 30, & 34 (“C1 Assay only” column). Table 8 also illustratessimilar trends across the sample conditions for each of the AHPV Assayconditions, with or without the presence of the C1 assays. DKA using alabeled detection probe comprising SEQ ID NO:15 provided a more robustsignal compared to using a labeled detection probe oligomer comprisingSEQ ID NO:71, though both were useful in these assays.

Conclusion: This example illustrates that linear detection probeoligomers are useful in assays for the detection of C1orf43 nucleicacids. Moreover, it is illustrated that these linear detection probeoligomers are useful in multiplexed nucleic acid amplification detectionassays, including multiplexed nucleic acid amplification and detectionassays wherein two of more of the target nucleic acid types aredifferentiated by the different probes.

Example 9—ThinPrep Sample Cellularity Control Testing

In this example, a cellularity control assay was performed to determinethe concentration of cells in cell collection media. Here, gynecologicalcell samples were collected at a number of clinical sites using cervicalsampling devices. The collected samples were immersed and rinsed inThinPrep vials filled with 20 mL of a cell preservative solution such asPreservCyt Solution (both the ThinPrep vial and the PreservCyt solutionare available from Hologic, Inc., Marlborough, Mass.). The ThinPrepsample vials were then capped and processed using a ThinPrep 5000processor, generally according to the instructions for use (Hologic,Inc.). ThinPrep sample processing began with a gentle dispersion step tomix the cell sample. The dispersed cell solutions were then captured toa gynecological ThinPrep Pap test filter to collect the cells. TheThinPrep 5000 processor monitors the rate of flow through the ThinPrepPap test filter during the collection process in order to prevent thecellular presentation from being too scant or too dense. Cell solutionsdetermined to be too scant, typically having less than 250 cells per mL,were marked as having insufficient cellular content. Sample solutionswith sufficient cellular content were transferred to a glass slide in a20 mm-diameter circle, and the slide was automatically deposited into afixative solution.

Sample Preparation: Seven hundred twenty two (722) cell sample solutionsdetermined to contain insufficient cellular content by the abovementioned processing step were then tested using a cellularity controlassay. The cellularity control assay was performed as generallydescribed in the above examples. To test the cell sample solutions, 1 mLof the solution was combined in a transport tube with 2.9 mL of sampletransport solution. An in vitro transcript (IVT) and C33A cells wereincluded for use as calibrator reactions. The IVT was prepared by serialdilution of a stock reagent to provide 10,000 copies/mL, 1,000copies/mL, and 100 copies/mL. The C33A cells were prepared by serialdilution of a stock cell solution to provide 2,500 cells/mL, 1,000cells/mL, and 100 cells/mL. The serial dilutions for each of the IVT andthe C33A calibrator reactions were at a 4 mL total volume.

Assay Set-Up: Oligonucleotides for capture, amplification and detectionof the ThinPrep cell samples included a target capture oligonucleotidecomprising the sequence of SEQ ID NO:12, a promoter primer comprisingthe sequence of SEQ ID NO:26, a non-T7 primer comprising the sequence ofSEQ ID NO:34, and a detectably labeled hairpin detection probecomprising the sequence of SEQ ID NO:14. The detection probe was labeledwith FAM/Dabcyl. Similarly, oligonucleotides were provided for thecapture, amplification and detection of the IVT control. Each reactioncondition was performed in real time. Assays were performed on a Panthersystem (Hologic, Inc.).

Results and Conclusions: Of the 722 tested samples, 702 samples weredetermined as valid (97.2%) by the Panther system. Samples were markedas invalid for a number of reasons, including bubbles in a reactionwell. All IVT and C33A reaction wells were determined as valid. Thevalid samples showed in this assay that 629 were positive (having atleast as low as 100 cells/mL) and 73 were negative (having a cell/mLcount below the limit of detection). Thus, the cellularity control assayworks well to determine the presence or absence of cells in a solution.The cellularity control assay is useful for determining the presence orabsence of gynecological cells suspended in a preservative solution,such as the PreservCyt solution. Further, the cellularity control isuseful as a reaction control, such as with the C33A control reactionsand/or as an assay for determining acceptance or rejection of a solutioncontaining collected cells.

TABLE OF SEQUENCESIn the following table, lower case letters indicate RNA and upper case letters indicate DNA. THS = target hybridizingsequence. X-C9 = -(CH₂)₉- linker follows position X. 5F3D = 5′-FAM, 3′-DABCYL labels.SEQ ID NO Description Sequence (5′ to 3′) 1C1orf43 (GenBank Accession No.GTGCGCGTGCCGCCTCGCCACGAGACACCTCTTTCCGGCTCCGCGAGTCCACCCCGCCTCCTTCACGGCGGCCCNM_015449.3)TGCCTCCACCACGTGACGCACGGATGGCCGCCGCTTCCTCTTACTGTCGTAGTTCCGCGTCTGAGCGCTCGACGCTCCTGGGTGCCATTGCCTGCCTGAGTCACGTGTCAGGGGGAAGCTGGAAGGCGTCGTTCTCCTTTCCCAGCTCTCCTGCCTGTCCGCCATGTTTTCAGGCCGGGTCTGGCTTGGTCTTCCCCCGTAAGGAAATGGCCGGGGAGCTCCAGGGGACCCAGGCGCCGTCGCTTCGGCGGAGCCTGGGCTGACCAGCCAGGACAGCGGGGTAAACCCGAACAATTCTGCGCGAGGTAGGGAGGCCATGGCGTCCGGCAGTAACTGGCTCTCCGGGGTGAATGTCGTGCTGGTGATGGCCTACGGGAGCCTGGACTTGAAAGAGGAGATTGATATTCGACTCTCCAGGGTTCAGGATATCAAGTATGAGCCCCAGCTCCTTGCAGATGATGATGCTAGACTACTACAACTGGAAACCCAGGGAAATCAAAGTTGCTACAACTATCTGTATAGGATGAAAGCTCTGGATGCCATTCGTACCTCTGAGATCCCATTTCATTCTGAAGGCCGGCATCCCCGTTCCTTAATGGGCAAGAATTTCCGCTCCTACCTGCTGGATCTGCGAAACACTAGTACGCCTTTCAAGGGTGTACGCAAAGCACTCATTGATACCCTTTTGGATGGCTATGAAACAGCCCGCTATGGGACAGGGGTCTTTGGCCAGAATGAGTACCTACGCTATCAGGAGGCCCTGAGTGAGCTGGCCACTGCGGTTAAAGCACGAATTGGGAGCTCTCAGCGACATCACCAGTCAGCAGCCAAAGACCTAACTCAGTCCCCTGAGGTCTCCCCAACAACCATCCAGGTGACATACCTCCCCTCCAGTCAGAAGAGTAAACGTGCCAAGCACTTCCTTGAATTGAAGAGCTTTAAGGATAACTATAACACATTGGAGAGTACTCTGTGACGGAGCTGAAGGACTCTTGCCGTAGATTAAGCCAGTCAGTTGCAATGTGCAAGACAGGCTGCTTGCCGGGCCGCCCTCGGAACATCTGGCCCAGCAGGCCCAGACTGTATCCATCCAAGTTCCCGTTGTATCCAGAGTTCTTAGAGCTTGTGTCTAAAGGGTAATTCCCCAACCCTTCCTTATGAGCATTTTTAGAACATTGGCTAAGACTATTTTCCCCCAGTAGCGCTTTTTTCTGGATTTGCATTCAGGTGTTATTCTTAATGTTTCTGTCAAAGCTTCTTAAAAATCTTCACTTGGTTTCAGCCATAGTTCACCTTCCCTGTTCCAGGTTTATTTAATTCCAAAGGTGAGAGTTGGAGTGAGATGTCTTCCATATCTATACCTTTGTGCACAGTTGAATGGGAACTGTTTGGGTTTAGGGCATCTTAGAGTTGATTGATGGAAAAAGCAGACAGGAACTGGTGGGAGGTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTGCTCCACTTAAACCAGATGTGTTGCAGCTTTCCTGACATGCAAGGATCTACTTTAATTCCACACTCTCATTAATAAATTGAATAAAAGGGAATGTTTTGGCACCTGATATAATCTGCCAGGCTATGTGACAGTAGGAAGGAATGGTTTCCCCTAACAAGCCCAATGCACTGGTCTGACTTTATAAATTATTTAATAAAATGAACTATTATCAAATAAAACGTATGAATCAGTCCTTTA 2 Not Used Not Used 3Construct for in vitro transcriptionCGACGAAGACTCTCTTTAATACGACTCACTATAGGGAAGCTTGTGCGCGTGCCGCCTCGCCACGAGACACCTCTwith T7 promoter (in bold)TTCCGGCTCCGCGAGTCCACCCCGCCTCCTTCACGGCGGCCCTGCCTCCACCACGTGACGCACGGATGGCCGCCGCTTCCTCTTACTGTCGTAGTTCCGCGTCTGAGCGCTCGACGCTCCTGGGTGCCATTGCCTGCCTGAGTCACGTGTCAGGGGGAAGCTGGAAGGCGTCGTTCTCCTTTCCCAGCTCTCCTGCCTGTCCGCCATGTTTTCAGGCCGGGTCTGGCTTGGTCTTCCCCCGTAAGGAAATGGCCGGGGAGCTCCAGGGGACCCAGGCGCCGTCGCTTCGGCGGAGCCTGGGCTGACCAGCCAGGACAGCGGGGTAAACCCGAACAATTCTGCGCGAGGTAGGGAGGCCATGGCGTCCGGCAGTAACTGGCTCTCCGGGGTGAATGTCGTGCTGGTGATGGCCTACGGGAGCCTGGACTTGAAAGAGGAGATTGATATTCGACTCTCCAGGGTTCAGGATATCAAGTATGAGCCCCAGCTCCTTGCAGATGATGATGCTAGACTACTACAACTGGAAACCCAGGGAAATCAAAGTTGCTACAACTATCTGTATAGGATGAAAGCTCTGGATGCCATTCGTACCTCTGAGATCCCATTTCATTCTGAAGGCCGGCATCCCCGTTCCTTAATGGGCAAGAATTTCCGCTCCTACCTGCTGGATCTGCGAAACACTAGTACGCCTTTCAAGGGTGTACGCAAAGCACTCATTGATACCCTTTTGGATGGCTATGAAACAGCCCGCTATGGGACAGGGGTCTTTGGCCAGAATGAGTACCTACGCTATCAGGAGGCCCTGAGTGAGCTGGCCACTGCGGTTAAAGCACGAATTGGGAGCTCTCAGCGACATCACCAGTCAGCAGCCAAAGACCTAACTCAGTCCCCTGAGGTCTCCCCAACAACCATCGCGGCCGCGTCCATCCAACACATGC 4 Capture oligomerCCAAACAGTTCCCATTCAACTGTGCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 5Capture oligomer THS CCAAACAGTTCCCATTCAACTGTGC 6 Capture oligomerCAAAGGUAUAGAUAUGGAAGACAUCUCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 7Capture oligomer THS CAAAGGUAUAGAUAUGGAAGACAUCUC 8 Capture oligomerCUGUGCACAAAGGUAUAGAUAUGGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 9Capture oligomer THS CUGUGCACAAAGGUAUAGAUAUGG 10 Capture oligomerCAUUCAACUGUGCACAAAGGUAUAGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 11Capture oligomer THS CAUUCAACUGUGCACAAAGGUAUAG 12 Capture oligomerCCAAACAGUUCCCAUUCAACUGUGCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 13Capture oligomer THS CCAAACAGUUCCCAUUCAACUGUGC 14C1orf43 molecular torch. 5F3D. GUGGAAUAACUUACCUUUGUGCccacRibonucleotides are 2′-O- methylated. 15 C1orf43 detection oligomer THSGUGGAAUAACUUACCUUUGUGC 16 C1orf43 molecular torch. 21-C9.GGUCAAGUGGGGAAGUUGGUGgacc 5F3D. Ribonucleotides are 2′-O- methylated. 17C1orf43 detection oligomer THS GGUCAAGUGGGGAAGUUGGUG 18C1orf43 molecular torch. 19-C9. GGGAAGUUGGUGAAUGUGGuccc5F3D. Ribonucleotides are 2′-O- methylated. 19C1orf43 detection oligomer THS GGGAAGUUGGUGAAUGUGG 20C1orf43 molecular torch. 5F3D. GGUGAAUGUGGAAUAACUUACCcaccRibonucleotides are 2′-O- methylated. 21 C1orf43 detection oligomer THSGGUGAAUGUGGAAUAACUUACC 22 c1orf43 molecular beacon. 5F3D.ggcucGUGGAAUAACUUACCUUUGUGCgagcc Ribonucleotides are 2′-O-methylated. 23C1orf43 detection oligomer THS GUGGAAUAACUUACCUUUGUGC 24C1orf43 molecular beacon. 5F3D. ggcucGGUGAAUGUGGAAUAACUUACCgagccRibonucleotides are 2′-O- methylated. 25 C1orf43 detection oligomer THSGGUGAAUGUGGAAUAACUUACC 26 Clorf43 promoter primerAATTTAATACGACTCACTATAGGGAGAGCATGTCAGGAAAGCTGCAACACATCTG 27C1orf43 amplification oligomer or GCATGTCAGGAAAGCTGCAACACATCTGamplification oligomer THS 28 Clorf43 promoter primerAATTTAATACGACTCACTATAGGGAGAGCTGCAACACATCTGGTTTAAGTGGAGC 29C1orf43 amplification oligomer or GCTGCAACACATCTGGTTTAAGTGGAGCamplification oligomer THS 30 Clorf43 promoter primerAATTTAATACGACTCACTATAGGGAGAGGAAAGCTGCAACACATCTGGTTTAAG 31C1orf43 amplification oligomer or GGAAAGCTGCAACACATCTGGTTTAAGamplification oligomer THS 32 Clorf43 promoter primerAATTTAATACGACTCACTATAGGGAGAGTAGATCCTTGCATGTCAGGAAAGCTGC 33C1orf43 amplification oligomer or GTAGATCCTTGCATGTCAGGAAAGCTGCamplification oligomer THS 34 C1orf43 amplification oligomer orGCAGACAGGAACTGGTGGGAG amplification oligomer THS 35C1orf43 amplification oligomer or GGCATCTTAGAGTTGATTGATGGamplification oligomer THS 36 C1orf43 amplification oligomer orCTTAGAGTTGATTGATGGAAAAAGC amplification oligomer THS 37C1orf43 amplification oligomer or GGAAAAAGCAGACAGGAACTGamplification oligomer THS 38 C1orf43 amplification oligomer orGCAGACAGGAACTGGTGG amplification oligomer THS 39Clorf43 region encompassingGAGATGTCTTCCATATCTATACCTTTGTGCACAGTTGAATGGGAACTGTTTGGGTTTAGGGCATCTTAGAGTTGexemplary ampliconsATTGATGGAAAAAGCAGACAGGAACTGGTGGGAGGTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTGCTCCACTTAAACCAGATGTGTTGCAGCTTTCCTGACATGCAAGGATCTAC 40Exemplary Clorf43 amplificationGGCATCTTAGAGTTGATTGATGGAAAAAGCAGACAGGAACTGGTGGGAGGTCAAGTGGGGAAGTTGGTGAATGTproductGGAATAACTTACCTTTGTGCTCCACTTAAACCAGATGTGTTGCAGCTTTCCTGACATGCAAGGATCTAC 41Exemplary Clorf43 amplificationGCAGACAGGAACTGGTGGGAGGTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTGCTCCACTTAproduct AACCAGATGTGTTGCAGCTTTCCTGACATGC 42Exemplary Clorf43 amplificationGCAGACAGGAACTGGTGGGAGGTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTGCTCCACTTAproduct AACCAGATGTGTTGCAGC 43 C1orf43 region encompassingAAAAAGCAGACAGGAACTGGTGGGAGGTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTGCTCCexemplary detection oligomer ACTTAAACCAGATGTGTT hybridization sites 44C1orf43 region encompassingTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTGCTCCACTTAAACexemplary detection oligomer hybridization sites 45C1orf43 region encompassing GTCAAGTGGGGAAGTTGGTGAATGTGGAATAACTTACCTTTGTexemplary detection oligomer hybridization sites 46C1orf43 region encompassingGGCATCTTAGAGTTGATTGATGGAAAAAGCAGACAGGAACTGGTGGGAGexemplary amplification oligomer (e.g., nonT7) hybridization sites 47exemplary amplification oligomer GCAGACAGGAACTG(e.g., nonT7) core sequence 48 exemplary amplification oligomerCTTAGAGTTGATTGATGG (e.g., nonT7) core sequence 49C1orf43 region encompassingGGUCAAGUGGGGAAGUUGGUGAAUGUGGAAUAACUUACCUUUGUGCexemplary detection oligomer hybridization sites 50exemplary detection oligomer core GUGGAAUAACUUACC seqeunce 51exemplary detection oligomer core GGGGAAGUUGGUG seqeunce 52C1orf43 region encompassingGTAGATCCTTGCATGTCAGGAAAGCTGCAACACATCTGGTTTAAGTGGAGCexemplary amplification oligomer (e.g., promoter primer)hybridization sites 53 Exemplary amplification oligomer GCTGCAACACATCTG(e.g., promoter primer) core sequence 54Exemplary amplification oligomer GGAAAGCTGC (e.g., promoter primer) coresequence 55 C1orf43 region encompassingCCAAACAGUUCCCAUUCAACUGUGCACAAAGGUAUAGAUAUGGAAGACAUCUCexemplary capture oligomer hybridization sites 56Exemplary capture oligomer core CAAAGGUAUAG sequence 57Exemplary capture oligomer core CAUUCAACUGUGC sequence 58Exemplary T7 promoter sequence AATTTAATACGACTCACTATAGGGAGA 59C1orf43 linear detection oligomer GUGAAUGUGGAAUAACUUAC 60C1orf43 linear detection oligomer GUGGGGAAGUUGGUG 61C1orf43 linear detection oligomer CACCUUAUUGAAUGGAAAC 62C1orf43 linear detection oligomer CUUCAACCACUUACACC 63C1orf43 linear detection oligomer CUUACACCUUAUUGAAUGG 64C1orf43 linear detection oligomer GAAUAACUUACCUUUGUG 65C1orf43 linear detection oligomer CAACCACUUACACCUUAUUG 66C1orf43 linear detection oligomer GAAGUUGGUGAAUGUG 67C1orf43 linear detection oligomer GUGGAAUAACUUACCUUUG 68C1orf43 linear detection oligomer GUGGGGAAGUUGGUGAAUG 69C1orf43 linear detection oligomer GAAUGUGGAAUAAC 70C1orf43 linear detection oligomer CUUACCUUUGUGCUCCAC 71C1orf43 linear detection oligomer GAAUAACUUACCUUUGUGCUC 72C1orf43 linear detection oligomer GUCAAGUGGGGAAGUUG

What is claimed is:
 1. A combination of oligomers comprising at leastfirst and second amplification oligomers, wherein the first and secondamplification oligomers are reverse and forward amplification oligomers,respectively; each comprise at least 10 nucleotides; and are configuredto specifically hybridize to first and second sites in the sequence ofSEQ ID NO: 39 and generate an amplicon therefrom, respectively.
 2. Amethod of detecting the presence or absence of a C1orf43 nucleic acid ina sample, comprising: contacting the sample with a combination ofoligomers comprising at least first and second amplification oligomers,performing a nucleic acid amplification reaction which produces at leasta first amplicon in the presence of the C1orf43 nucleic acid, anddetecting the presence or absence of the first amplicon, wherein: thefirst amplicon is produced through extension of the first and secondamplification oligomers in the presence of the C1orf43 nucleic acid; andwherein the first and second amplification oligomers are reverse andforward amplification oligomers, respectively; each comprise at least 10nucleotides; and are configured to specifically hybridize to first andsecond sites in the sequence of SEQ ID NO: 39, respectively.
 3. Thecombination of claim 1 or method of claim 2, wherein at least one of theamplification oligomers is a promoter-primer.
 4. The combination ormethod of any one of the preceding claims, wherein the firstamplification oligomer is a promoter-primer.
 5. The combination ormethod of any one of claims 3-4, wherein the promoter-primer comprises aT7 promoter which is located 5′ of a target-hybridizing sequence.
 6. Thecombination or method of claim 5, wherein the T7 promoter comprises thesequence of SEQ ID NO:
 58. 7. The combination or method of any one ofthe preceding claims, wherein the first and second amplificationoligomer are configured to generate an amplicon comprising at least 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 144 nucleotides ofthe sequence of SEQ ID NO:
 40. 8. The combination or method of claim 7,wherein the first and second amplification oligomer are configured togenerate an amplicon comprising the sequence of SEQ ID NO:
 41. 9. Thecombination or method of claim 7, wherein the first and secondamplification oligomer are configured to generate an amplicon comprisingthe sequence of SEQ ID NO:
 42. 10. The combination or method of any oneof the preceding claims, wherein the first amplification oligomer isconfigured to specifically hybridize to a first site in the complementof SEQ ID NO:
 52. 11. The combination or method of claim 10, wherein thefirst site comprises the sequence of SEQ ID NO:
 53. 12. The combinationor method of claim 10, wherein the first site comprises the sequence ofSEQ ID NO:
 54. 13. The combination or method of any one of the precedingclaims, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 27,optionally with up to two mismatches.
 14. The combination or method ofany one of claims 1-12, wherein the first amplification oligomercomprises a target-hybridizing sequence comprising the sequence of SEQID NO: 29, optionally with up to two mismatches.
 15. The combination ormethod of any one of claims 1-12, wherein the first amplificationoligomer comprises a target-hybridizing sequence comprising the sequenceof SEQ ID NO: 31, optionally with up to two mismatches.
 16. Thecombination or method of any one of claims 1-12, wherein the firstamplification oligomer comprises a target-hybridizing sequencecomprising the sequence of SEQ ID NO: 33, optionally with up to twomismatches.
 17. The combination or method of any one of the precedingclaims, wherein the first amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 27,29, 31, or
 33. 18. The combination or method of any one of the precedingclaims, wherein the first amplification oligomer comprises the sequenceof SEQ ID NO: 26, 28, 30, or
 32. 19. The combination or method of anyone of the preceding claims, wherein the second amplification oligomeris configured to specifically hybridize to a second site in thecomplement of SEQ ID NO:
 46. 20. The combination or method of claim 19,wherein the second site comprises the sequence of SEQ ID NO:
 47. 21. Thecombination or method of claim 19, wherein the second site comprises thesequence of SEQ ID NO:
 48. 22. The combination or method of any one ofthe preceding claims, wherein the second amplification oligomercomprises a target-hybridizing sequence comprising the sequence of SEQID NO: 34, optionally with up to two mismatches.
 23. The combination ormethod of any one of claims 1-21, wherein the second amplificationoligomer comprises a target-hybridizing sequence comprising the sequenceof SEQ ID NO: 35, optionally with up to two mismatches.
 24. Thecombination or method of any one of claims 1-21, wherein the secondamplification oligomer comprises a target-hybridizing sequencecomprising the sequence of SEQ ID NO: 36, optionally with up to twomismatches.
 25. The combination or method of any one of claims 1-21,wherein the second amplification oligomer comprises a target-hybridizingsequence comprising the sequence of SEQ ID NO: 37, optionally with up totwo mismatches.
 26. The combination or method of any one of claims 1-21,wherein the second amplification oligomer comprises a target-hybridizingsequence comprising the sequence of SEQ ID NO: 38, optionally with up totwo mismatches.
 27. The combination or method of any one of thepreceding claims, wherein the second amplification oligomer comprises atarget-hybridizing sequence comprising the sequence of SEQ ID NO: 34,35, 36, 37, or
 38. 28. The combination or method of any one of thepreceding claims, wherein the combination further comprises at least oneprobe oligomer that comprises at least 10 nucleotides and is configuredto specifically hybridize to an amplicon produced from the first andsecond amplification oligomers.
 29. The combination or method of claim28, wherein the probe oligomer is configured to specifically hybridizeto a detection site in a nucleic acid having the sequence of SEQ ID NO:43.
 30. A probe oligomer that comprises at least 10 nucleotides and isconfigured to specifically hybridize to a detection site in a nucleicacid having the sequence of SEQ ID NO:
 43. 31. The combination, method,or probe oligomer of any one of claims 28-30, wherein the probe oligomeris configured to specifically hybridize to a detection site in thesequence of SEQ ID NO:
 44. 32. The combination, method, or probeoligomer of any one of claims 28-30, wherein the probe oligomer isconfigured to specifically hybridize to a detection site in a nucleicacid having the sequence of SEQ ID NO:
 45. 33. The combination, method,or probe oligomer of any one of claims 28-30, wherein the probe oligomeris configured to specifically hybridize to a detection site in a nucleicacid having the sequence of SEQ ID NO:
 49. 34. The combination, method,or probe oligomer of claim 33, wherein the probe oligomer comprises atarget hybridizing sequence comprising the sequence of SEQ ID NO: 50.35. The combination, method, or probe oligomer of claim 33, wherein theprobe oligomer comprises a target hybridizing sequence comprising thesequence of SEQ ID NO:
 51. 36. The combination, method, or probeoligomer of any one of claims 28-35, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 15 with up to two mismatches.
 37. The combination, method, orprobe oligomer of any one of claims 28-35, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 17 with up to two mismatches.
 38. The combination, method, orprobe oligomer of any one of claims 28-35, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 19 with up to two mismatches.
 39. The combination, method, orprobe oligomer of any one of claims 28-35, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 21 with up to two mismatches.
 40. The combination, method, orprobe oligomer of any one of claims 28-35, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 23 with up to two mismatches.
 41. The combination, method, orprobe oligomer of any one of claims 28-35, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 25 with up to two mismatches.
 42. The combination, method, orprobe oligomer of any one of claims 28-41, wherein the probe oligomercomprises a target hybridizing sequence comprising the sequence of SEQID NO: 15, 17, 19, 21, 23, or
 25. 43. The combination, method, or probeoligomer of any one of claims 28-41, wherein the probe oligomercomprises the sequence of SEQ ID NO: 14, 16, 18, 20, 22, or
 24. 44. Thecombination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 59 with up to two mismatches. 45.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 60 with up to two mismatches. 46.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 64 with up to two mismatches. 47.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 66 with up to two mismatches. 48.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 67 with up to two mismatches. 49.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 68 with up to two mismatches. 50.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 69 with up to two mismatches. 51.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 70 with up to two mismatches. 52.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 71 with up to two mismatches. 53.The combination, method, or probe oligomer of any one of claims 28-35,wherein the probe oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO: 72 with up to two mismatches. 54.A probe oligomer that comprises a target hybridizing sequence comprisingthe sequence of any one of SEQ ID NO: 61, 62, 63, or 65 with up to twomismatches
 55. The combination, method, or probe oligomer of any one ofclaim 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 61 with up totwo mismatches.
 56. The combination, method, or probe oligomer of anyone of claim 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 62 with up totwo mismatches.
 57. The combination, method, or probe oligomer of anyone of claim 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 63 with up totwo mismatches.
 58. The combination, method, or probe oligomer of anyone of claim 28 or 54, wherein the probe oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO: 65 with up totwo mismatches.
 59. The combination, method, or probe oligomer of anyone of claim 28-35 or 44-58, wherein the probe oligomer comprises atarget hybridizing sequence comprising the sequence of any one of SEQ IDNOs: 59-72.
 60. The combination, method, or probe oligomer of any one ofclaims 28-59, wherein the probe oligomer comprises 2′-O-methyl-ribose inits backbone.
 61. The combination, method, or probe oligomer of claim60, wherein at least half, at least 90%, or all of the sugars in theprobe oligomer are 2′-O-methyl-ribose.
 62. The combination or method ofany one of claims 1-27, wherein one or more of the first and secondamplification oligomers comprises a non-nucleotide detectable label. 63.The combination, method, or probe oligomer of any one of claims 28-61,wherein at least one probe oligomer comprises a non-nucleotidedetectable label.
 64. The combination, method, or probe oligomer of anyone of claims 62-63, wherein the non-nucleotide detectable label is afluorescent label.
 65. The combination, method, or probe oligomer ofclaim 63 or 64, wherein the probe oligomer comprises a quencher.
 66. Thecombination, method, or probe oligomer of claim 65, wherein thenon-nucleotide detectable label is a fluorescent label and the quencherabsorbs fluorescence to a greater extent when the probe is free thanwhen the probe is annealed to a target nucleic acid.
 67. Thecombination, method, or probe oligomer of any one of claims 64-66,wherein the fluorescent label is FAM, HEX, or acridine.
 68. Thecombination, method, or probe oligomer of any one of claims 65-67,wherein the quencher is DABCYL or ROX.
 69. The combination, method, orprobe oligomer of any one of claims 65-68, wherein the fluorescent labelis attached to the 5′-terminus of the probe oligomer and the quencher isattached to the 3′-terminus of the probe oligomer, or the fluorescentlabel is attached to the 3′-terminus of the probe oligomer and thequencher is attached to the 5′-terminus of the probe oligomer.
 70. Thecombination, method, or probe oligomer of any one of claims 62-63,wherein the non-nucleotide detectable label is a chemiluminescent label.71. The combination, method, or probe oligomer of any one of claim 28-61or 63-70, wherein the probe oligomer comprises a firstself-complementary region at its 5′ end and a second self-complementaryregion at its 3′ end.
 72. The combination, method, or probe oligomer ofclaim 71, wherein the self-complementary regions can hybridize to form 3to 7 Watson-Crick or wobble base pairs.
 73. The combination, method, orprobe oligomer of claim 71, wherein the self-complementary regions canhybridize to form 4 Watson-Crick or wobble base pairs.
 74. Thecombination, method, or probe oligomer of any one of claims 63-71,wherein the probe oligomer is a linear probe oligomer.
 75. A method ofdetecting the presence or absence of a C1orf43 nucleic acid in a sample,comprising: contacting the sample with the probe oligomer of any one ofclaim 30-61 or 63-74; performing a hybridization reaction which producesa complex of the probe oligomer and the C1orf43 nucleic acid in thepresence of the C1orf43 nucleic acid; and detecting the presence orabsence of the complex of the probe oligomer and the C1orf43 nucleicacid.
 76. The method of claim 75, wherein the hybridization reaction isa hybridization protection assay.
 77. The method of claim 75 or 76,wherein the hybridization reaction is a dual kinetic assay.
 78. Themethod of any one of claims 75-77, wherein the probe oligomer functionsas a flasher probe in the dual kinetic assay.
 79. The method of any oneof claims 75-77, wherein the probe oligomer functions as a glower probein the dual kinetic assay.
 80. The method of any one of claims 75-79,wherein the C1orf43 nucleic acid comprises a C1orf43 amplicon.
 81. Themethod of any one of claims 75-79, wherein the C1orf43 nucleic acidcomprises C1orf43 RNA from cells in the sample.
 82. The method of anyone of claims 75-81, wherein the sample is contacted with a combinationof oligomers comprising the probe oligomer.
 83. The combination ormethod of any one of claim 1-29, 31-56, or 82, wherein the combinationfurther comprises at least one capture oligomer that comprises at least10 nucleotides and is configured to specifically hybridize to a capturesite in a C1orf43 nucleic acid.
 84. The combination or method of claim83, wherein the capture site is in the sequence of SEQ ID NO: 1, 2, or3.
 85. The combination or method of claim 83, wherein the capture siteis in the sequence of SEQ ID NO:
 39. 86. A method of isolating C1orf43nucleic acid from a sample, comprising: contacting the sample with atleast one capture oligomer under conditions permissive for forming oneor more complexes of a capture oligomer and the C1orf43 nucleic acid,thereby forming a composition, wherein the capture oligomer comprises atleast 10 nucleotides and is configured to specifically hybridize to acapture site in the sequence of SEQ ID NO: 39; and isolating the captureoligomer from the composition.
 87. The method of claim 86, whereinisolating the capture oligomers comprises associating the captureoligomers with a solid support, and washing the solid support.
 88. Themethod of claim 87, wherein the solid support comprises a poly-Nsequence that is complementary to a portion of the capture oligomer. 89.The method of claim 87, wherein the solid support comprises a bindingagent that recognizes an affinity tag present in the capture oligomer.90. A capture oligomer that comprises at least 10 nucleotides and isconfigured to specifically hybridize to a capture site in the sequenceof SEQ ID NO:
 39. 91. The combination, method, or capture oligomer ofany one of claims 83-90, wherein the capture site is in the sequence ofSEQ ID NO:
 55. 92. The combination, method, or capture oligomer of anyone of claims 83-90, wherein the capture oligomer comprises a targethybridizing sequence comprising the sequence of SEQ ID NO:
 56. 93. Thecombination, method, or capture oligomer of any one of claims 83-90,wherein the capture oligomer comprises a target hybridizing sequencecomprising the sequence of SEQ ID NO:
 57. 94. The combination, method,or capture oligomer of any one of claims 83-93, wherein the captureoligomer comprises a target hybridizing sequence comprising the sequenceof SEQ ID NO: 5 with up to two mismatches.
 95. The combination, method,or capture oligomer of any one of claims 83-93, wherein the captureoligomer comprises a target hybridizing sequence comprising the sequenceof SEQ ID NO: 7 with up to two mismatches.
 96. The combination, method,or capture oligomer of any one of claims 83-93, wherein the captureoligomer comprises a target hybridizing sequence comprising the sequenceof SEQ ID NO: 9 with up to two mismatches.
 97. The combination, method,or capture oligomer of any one of claims 83-93, wherein the captureoligomer comprises a target hybridizing sequence comprising the sequenceof SEQ ID NO: 11 with up to two mismatches.
 98. The combination, method,or capture oligomer of any one of claims 83-93, wherein the captureoligomer comprises a target hybridizing sequence comprising the sequenceof SEQ ID NO: 13 with up to two mismatches.
 99. The combination, method,or capture oligomer of any one of claims 83-93, wherein the captureoligomer comprises a target hybridizing sequence comprising the sequenceof SEQ ID NO: 5, 7, 9, 11, or
 13. 100. The combination, method, orcapture oligomer of any one of claims 83-99, wherein the captureoligomer comprises the sequence of SEQ ID NO: 4, 6, 8, 10, or
 12. 101.The combination, method, or capture oligomer of any one of claims83-100, wherein the capture oligomer comprises 2′-O-methyl-ribose in itsbackbone.
 102. The combination, method, or capture oligomer of claim101, wherein at least half, at least 90%, or all of the sugars in thetarget hybridizing sequence of the capture oligomer are2′-O-methyl-ribose.
 103. The combination, method, or capture oligomer ofany one of claims 83-102, wherein the capture oligomer further comprisesa non-nucleotide affinity label.
 104. The combination, method, orcapture oligomer of any one of claims 83-102, wherein the captureoligomer further comprises a non-C1orf43 sequence.
 105. The combination,method, or capture oligomer of claim 104, wherein the non-C1orf43sequence is a poly-N sequence.
 106. The combination, method, or captureoligomer of claim 105, wherein the poly-N sequence is a poly-A or poly-Tsequence.
 107. A combination comprising the capture oligomer accordingto any one of claims 90-106 and one or more amplification oligomers,wherein the amplification oligomer is configured to specificallyhybridize to a site in the sequence of SEQ ID NO:
 39. 108. Thecombination of claim 107, wherein the one or more amplificationoligomers includes the first amplification oligomer as recited in anyone of claims 4-18.
 109. The combination of claim 107 or 108, whereinthe one or more amplification oligomers includes the secondamplification oligomer as recited in any one of claims 19-27.
 110. Thecombination of any one of claims 107-109, further comprising the probeoligomer as recited in any one of claim 28-61 or 63-74.
 111. The methodof any one of claim 86-89 or 91-106, further comprising performing alinear amplification wherein at least one amplification oligomer isextended.
 112. The method of claim 111, wherein prior to the linearamplification, the amplification oligomer is associated with a complexof C1orf43 nucleic acid and a capture oligomer and the complex isassociated with a solid support, and the method comprises washing thesolid support.
 113. The method of claim 112, wherein the solid supportis a population of microbeads.
 114. The method of claim 113, wherein themicrobeads of the population are magnetic.
 115. The method of any one ofclaims 112-114, wherein following the washing step, the method comprisesadding one or more additional amplification oligomers oppositelyoriented to an amplification oligomer associated with the complex ofC1orf43 nucleic acid and the capture oligomer.
 116. The method of claim115, wherein the one or more oppositely oriented additionalamplification oligomers includes a promoter-primer.
 117. The method ofclaim 116, wherein the one or more oppositely oriented additionalamplification oligomers includes an oligomer that is not apromoter-primer.
 118. The method of any one of claims 115-117, whereinthe one or more oppositely oriented additional amplification oligomersincludes the second amplification oligomer as recited in any one ofclaims 19-27.
 119. The method of any one of claims 115-118, furthercomprising performing an exponential amplification following the linearamplification.
 120. The method of claim 119, wherein the exponentialamplification is transcription-mediated amplification.
 121. The methodof any one of claim 2-29, 31-89, 91-105, or 111-120, further comprisingquantifying C1orf43 nucleic acid in the sample.
 122. A kit orcomposition comprising at least one, two, three, or four of a firstamplification oligomer, a second amplification oligomer, a probeoligomer, or a capture oligomer recited in any one of the precedingclaims.
 123. The kit or composition of claim 122, comprising at leastone probe oligomer as recited in any one of claim 28-61 or 63-74. 124.The kit or composition of any one of claims 122-123, comprising at leastone capture oligomer as recited in any one of claim 83-85 or 90-106.125. The kit or composition of any one of claims 122-124, comprising thefirst amplification oligomer as recited in any one of claims 4-18 andthe second amplification oligomer as recited in any one of claims 19-27.126. A kit according to any one of claims 122-125 or comprising thecombination of any one of claim 1, 3-62, 63-85, or 91-110.
 127. Acomposition according to any one of claims 122-125 or comprising thecombination of any one of claim 1, 3-62, 63-85, or 91-110.
 128. Thecomposition of claim 127, which is aqueous, frozen, or lyophilized. 129.Use of the combination, method, composition, capture oligomer, probeoligomer, or kit of any one of the preceding claims for detecting thepresence or absence of a C1orf43 nucleic acid in a sample.
 130. Thecombination, method, composition, capture oligomer, probe oligomer, orkit of any one of claims 1-128, for use in detecting the presence orabsence of a C1orf43 nucleic acid in a sample.
 131. Use of thecombination, method, composition, capture oligomer, probe oligomer, orkit of any one of claims 1-128 for quantifying a C1orf43 nucleic acid ina sample.
 132. The combination, method, composition, capture oligomer,probe oligomer, or kit of any one of claims 1-128, for use inquantifying a C1orf43 nucleic acid in a sample.
 133. The use,combination, method, composition, capture oligomer, probe oligomer, orkit of any one of claim 2-29, 31-53, 55-89, 91-106, 111-121, or 126-132,wherein the sample comprises human mRNA.
 134. The use, combination,method, composition, or kit of claim 133, wherein the human mRNAcomprises mRNA from bladder, ductus deferens, epididymis, kidney, lymphnode, pancreas, peripheral blood lymphocytes, penis, prostate, seminalvesicle, or spleen.
 135. The use, combination, method, composition, orkit of claim 133, wherein the human mRNA comprises mRNA from a vaginalor cervical sample.
 136. The use, combination, method, composition, orkit of claim 135, wherein the vaginal or cervical sample is a vaginal orcervical swab.
 137. The use, combination, method, composition, or kit ofany one of claim 2-29, 31-53, 55-89, 91-106, 111-121, or 126-136,wherein the method or use further comprises detecting the presence orabsence of at least one nucleic acid of a microbe or pathogen.
 138. Theuse, combination, method, composition, or kit of claim 137, wherein theat least one nucleic acid of a microbe or pathogen comprises humanpapillomavirus nucleic acid, Chlamydia trachomatis nucleic acid,Neisseria gonorrheae nucleic acid, Trichomonas vaginalis nucleic acid,or Mycoplasma genitalium nucleic acid.
 139. The use, combination,method, composition, or kit of any one of claim 2-29, 31-53, 55-89,91-106, 111-121, or 126-138, wherein the method or use further comprisesdetecting the presence or absence of at least one mRNA other thanC1orf43.
 140. The use, combination, method, composition, or kit of claim139, wherein the mRNA other than C1orf43 is a human mRNA other thanC1orf43.